Insect inhibitory lipid acyl hydrolases

ABSTRACT

The present invention discloses DNA sequences encoding plant and novel lipid acyl hydrolase proteins having coleopteran specific insect inhibitory activity, as well as variants and permuteins having enhanced levels of activity directed to controlling coleotperan insect infestation and enhanced levels of expression in planta. Additionally, catalytic dyad active site conformation is disclosed for both dicot and monocot plant derived non-specific lipid acyl hydrolases having coleopteran insect inhibitory properties.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U.S. Provisional ApplicationsSerial No. 60/174,669 filed Jan. 6, 2000 and 60/219,912 filed Jul. 21,2000, and claims the benefit of priority thereto.

FIELD OF THE INVENTION

[0002] The invention relates to the design, preparation, and use ofpatatin and structurally related proteins which have insect inhibitoryproperties and which display a requirement for catalysis structuredaround an active site catalytic dyad. Patatin and related proteinsinclude amino acid sequence variants which maintain the active sitecatalytic dyad motif and which maintain insect inhibitory properties noless than the native protein, and include permuteins which have hadtheir amino acid sequences rearranged at at least one breakpoint.

BACKGROUND OF THE INVENTION

[0003] The use of natural products, including proteins, is a well knownmethod of controlling many insect, fungal, viral, bacterial, andnematode pathogens. For example, Sendotoxin proteins of Bacillusthuringiensis (B.t.) are used to control both lepidopteran andcoleopteran insect pests. Genes producing these proteins have beenintroduced into and expressed by various plants, including cotton,tobacco, corn, wheat, rice, potato, and tomato, a number of differentvarieties of forage and turf grasses, ornamental flowers, and otherfruit and vegetable crops. There are, however, several economicallyimportant insect pests that are not particularly susceptible to B.t.endotoxins. Examples of such important pests are the boll weevil (BWV),Anthonomus grandis, and corn rootworm (CRW), Diabrotica spp. Inaddition, having other, different gene products which do not functionlike Bt proteins for control of insects which are susceptible to B.t.endotoxins is important, if not vital, for effective and long termresistance management practices.

[0004] Recently, alternative species of bacteria have been identifiedwhich are capable of producing proteins displaying insect inhibitoryeffects. Photorhabdus and Xenorhabdus comprise broad genus' of bacteriawhich occupy the gut of entomopathogenic nematodes. upon invasion of theinsect body by the nematode, the entomopathogenic bacteria are releasedfrom the gut of the nematode into the insect haemolymph where theyproliferate, inhibit further development of the insect, and produce anutrient enriched monoculture designed specifically for symbioticnematode and bacterial survival. A variety of extracellular proteins areproduced by these bacterial symbionts, each insect inhibitory proteinhaving distinct insect genus and species specificity, each proteinlikely being structurally and probably functionally different from BTICP's. (Ensign et al., Insecticidal Protein Toxins from Photorhabdus, WO97/17432; Jarrett et al., Pesticidal Agents, WO 98/08388;Ffrench-Constant et al., Novel insecticidal Toxins fromNematode-Symbiotic Bacteria, Cellular and Molecular Life Sciences57:828-833, May 2000).

[0005] Plant proteins have also been identified which exhibit insectinhibitory effects.

[0006] One such protein is patatin, a non-specific lipid acyl hydrolase,which is the major storage protein of potato tubers (Gaillaird, T.,Biochem. J. 121: 379-390, 1971; Racusen, D., Can. J. Bot., 62:1640-1644, 1984; Andrews, D. L., et al., Biochem. J., 252: 199-206,1988). Patatin has been shown to control various insects, includingwestern rootworm (WCRW, Diabrotica virigifera), southern corn rootworm(SCRW, Diabrotica undecimpunctata), and boll weevil (BWV, Anthonomusgrandis) (U.S. Pat. No. 5,743,477, issued Apr. 28, 1998). Patatinrelated protein sequences have been identified in a variety of plantspecies. When applied at an appropriate level in artificial diet, potatopatatin is lethal to some larvae and will stunt the growth of survivorsso that maturation is prevented or severely delayed, resulting in noreproduction. These proteins display non-specific lipid acyl hydrolaseactivity. Studies have shown that the enzyme activity is essential forits insect inhibitory activity (Strickland, J. A., et al., PlantPhysiol., 109: 667-674, 1995). Patatins may be applied directly to theplants or introduced in other ways well known in the art, such asthrough the application of plant-colonizing microorganisms, which havebeen transformed to produce the enzymes, or by the plants themselvesafter similar transformation.

[0007] In potato, the patatins are found predominantly in tubers, butalso at much lower levels in other plant organs (Hofgen, R. andWillmitzer, L., Plant Science, 66: 221-230, 1990). Genes that encodepatatins have been previously isolated by Mignery, G. A., et al.(Nucleic Acids Research, 12: 7987-8000, 1984; Mignery, G. A., et al.,Gene, 62: 27-44, 1988; Stiekema, et al., Plant Mol. Biol., 11: 255-269,1988) and others. Patatins are found in other plants, particularlysolanaceous species (Ganal, et al., Mol. Gen. Genetics, 225: 501-509,1991; Vancanneyt, et al., Plant Cell, 1: 533-540, 1989) and recently Zeamays (Patent number WO 96/37615). Rosahl, et al. (EMBO J., 6: 1155-1159,1987) transferred a patatin coding sequence into tobacco plants, andobserved expression of patatin, demonstrating that patatin can beheterologously expressed by plants. Modification of coding sequences hasbeen demonstrated to improve expression of other insect inhibitoryprotein genes such as the δ-endotoxin sequences from Bacillusthuringiensis (Fischhoff and Perlak; WO 93/07278). However, expressionof a native plant species sequence encoding a protein exhibiting insectinhibitory properties in a plant at levels not previously observed innature would be particularly advantageous. Such sequences would notrequire coding sequence modifications found to be necessary to achievesubstantial levels of insect protection as have been required forsequences encoding Bt proteins for example.

[0008] As indicated above, plant non-specific lipid acyl hydrolases havebeen identified from a variety of plant sources including potato tubers.Speculation on the role of the enzyme has been centered on theirinvolvement in the turnover of membrane lipids, however one reportidentified an serine residue required for hydrolase activity andconserved sequence flanking the residue in potato patatin based oninactivation of the enzyme acyl lipid hydrolase activity when treatedwith diisopropyl fluorophosphate and an amino acid sequence alignmentwith a patatin isoform (Walsh et al., U.S. Pat. No. 5,743,477; Apr. 28,1998). Based on the amino acid sequence of potato patatin, Walsh et al.proposed that Ser-77 in the hydrolase motif, Gly-X-Ser-X-Gly is thecatalytic residue required for enzyme function as well as insectinhibitory activity.

[0009] The inventors herein have identified a patatin isozyme designatedPat17, and used alanine scanning mutagenesis and X-ray crystallographyto solve the structure of the patatin enzyme and to identify additionalresidues responsible for both catalytic activity and insect inhibitorybioactivity.

[0010] Novel proteins generated by the method of sequence transpositionresembles that of naturally occurring pairs of proteins that are relatedby linear reorganization of their amino acid sequences (Cunningham, etal. Proc. Natl. Sci., U.S.A., 76: 3218-3222, 1979; Teather, et al., J.Bacteriol., 172: 3837-3841, 1990; Schimming, et al., Eur. J. Biochem.,204: 13-19, 1992; Yamiuchi, et al., FEBS Lett., 260: 127-130, 1991;MacGregor, et al., FEBS. Lett., 378: 263-266, 1996). The first in vitroapplication of sequence rearrangement to proteins was described byGoldenberg and Creighton (Goldenberg and Creighton, J. Mol. Biol., 165:407-413, 1983). A new N-terminus is selected at an internal site(breakpoint) of the original sequence, the new sequence having the sameorder of amino acids as the original from the breakpoint until itreaches an amino acid that is at or near the original C-terminus. Atthis point the new sequence is joined, either directly or through anadditional portion or sequence (linker), to an amino acid that is at ornear the original N-terminus, and the new sequence continues with thesame sequence as the original until it reaches a point that is at ornear at or near the amino acid that was N-terminal to the breakpointsite of the original sequence, this residue forming the new C-terminusof the chain. This approach has been applied to proteins which range insize from 58 to 462 amino acids and represent a broad range ofstructural classes (Goldenberg and Creighton, J. Mol. Biol., 165:407413, 1983; Li and Coffino, Mol. Cell. Biol., 13: 2377-2383, 1993;Zhang, et al., Nature Struct. Biol., 1: 434-438, 1995; Buchwalder, etal., Biochemistry, 31: 1621-1630, 1994; Protasova, et al., Prot. Eng.,7: 1373-1377, 1995; Mullins, et al., J. Am. Chem. Soc., 116: 5529-5533,1994; Garrett, et al., Protein Science, 5: 204-211, 1996; Hahn, et al.,Proc. Natl. Acad. Sci. U.S.A., 91: 10417-10421, 1994; Yang andSchachman, Proc. Natl. Acad. Sci. U.S.A., 90: 11980-11984, 1993; Luger,et al., Science, 243: 206-210, 1989; Luger, et al., Prot. Eng., 3:249-258, 1990; Lin, et al., Protein Science, 4: 159-166, 1995; Vignais,et al., Protein Science, 4: 994-1000, 1995; Ritco-Vonsovici, et al.,Biochemistry, 34: 16543-16551, 1995; Horlick, et al., Protein Eng., 5:427431, 1992; Kreitman, et al., Cytokine, 7: 311-318, 1995; Viguera, etal., Mol. Biol., 247: 670-681, 1995; Koebnik and Kramer, J. Mol. Biol.,250: 617-626, 1995; Kreitman, et al., Proc. Natl. Acad. Sci., 91:6889-6893, 1994).

[0011] Thus, there exists a need to identify novel protein sequenceswhich are insect inhibitory, which are not related to Bt insectinhibitory proteins in form or function, and which are safe forexpression in human and animal food supplies. Such proteins should havemodes of action distinct from those of Bt insect inhibitory proteins orXenorhabdus or Photorhabdus insect inhibitory proteins and should actsynergistically with BT's or Xenorhabdus or Photorhabdus insectinhibitory proteins to aid in preventing the onset of insect speciesresistance developed in response to providing only single insectinhibitory proteins in compositions of matter as food sources topopulations of insects in fields of recombinant crops.

SUMMARY OF THE INVENTION

[0012] The present invention provides a method for identifying a lipidacyl hydrolase having insect inhibitory properties comprising isolatingand purifying a protein having lipid acyl hydrolase activity; obtaininga three dimensional crystal structure of said protein; and identifyingthe amino acid sequence of said protein; wherein said amino acidsequence contains a serine active site motif gly-xxx-ser-xxx-gly and anaspartate active site motif glu-xxx-xxx-leu-val-asp-gly. Modificationsof these motifs should disrupt the hydrolase and the insect inhibitoryproperties of the protein.

[0013] Furthermore, the invention provides a method of inhibiting insectinfestation of a plant or plant part comprising providing in theinsect's plant diet an insect inhibitory effective amount of a lipidacyl hydrolase having insect inhibitory properties when ingested by saidinsect, wherein the amino acid sequence of said hydrolase comprises aserine active site motif gly-xxx-ser-xxx-gly and an aspartate activesite motif glu-xxx-xxx-leu-val-asp-gly. The serine active site motif canbe shown to be required by treating the hydrolase with a substrate whichbinds specifically and irreversibly to the serine in the serine activesite motif, such as diisopropyl fluorophosphate. The serine active sitemotif and/or the aspartate active site motif can be shown to be requiredby modifying the amino acid sequence within each motif to show loss offunction of hydrolase and insect inhibition.

[0014] The invention further provides a method for protecting a plant orpart thereof against insect infestation comprising providing an insectcontrolling amount of a plant lipid acyl hydrolase protein having acrystal structure containing a serine active site motif G-X-S-X-G and anaspartate active site motif E-X-X-L-V-D-G, each motif being present inthe active site cleft defined by the crystal structure and the serineand aspartate residues in each motif being required for the catalyticfunction of the hydrolase, and the catalytic function of the hydrolasebeing required for functional and effective insect inhibition whenprovided in diet form to a susceptible insect larvae.

[0015] Novel protein sequences having lipid acyl hydrolase activity, aswell as nucleic acid sequences encoding said protein sequences aredisclosed. The proteins maintain desirable insect inhibitory propertieswhen expressed in plants.

[0016] Alanine scanning and ‘rational substitution’ is performed onidentified peptide sequences to determine specific amino acids whichcontribute to lipid acyl hydrolase activity. Individual mutations areintroduced into the whole protein sequence by methods such as sitedirected mutagenesis of the encoding nucleic acid sequence.

[0017] Permuteins of the novel protein sequences may be constructed toreduce or eliminate allergenic properties or to improve proteinstability and protein expression. The encoding nucleic acid sequence ismodified to produce a protein with a re-arranged amino acid sequence,while maintaining insect inhibitory properties.

[0018] The novel proteins may be used in controlling insects, asnutritional supplements, in immunotherapy protocols, and in otherpotential applications. Transgenic plant cells and plants containing theencoding nucleic acid sequence may be particularly beneficial in thecontrol of insects, and as a nutritional/immunotherapy material.

[0019] One object of the present invention is to provide a method forprotecting a plant or plant part from insect infestation.

[0020] Another object of the present invention is to provide a methodfor identifying a lipid acyl hydrolase enzyme which functions to inhibitinsect infestation. The method consists of identifying a proteindisplaying lipid acyl hydrolase activity. A DNA sequence encoding theprotein sequence can either be synthesized by back-translating the aminoacid sequence, or by identifying a DNA coding sequence from a sourcefrom which the enzyme was isolated and purified. The enzyme can betreated with diisopropyl fluorophosphate to identify a serine residueinvolved in lipid acyl hydrolase activity. The crystal structure of theenzyme can then be determined, and the three dimensional model of thestructure can be used to identify the active site and additionalresidues involved in active site catalysis. Other residues, such asHis109 exemplified in Pat17, can be identified which are crucial forenzyme stability using alanine scanning mutagenesis. An enzymedisplaying lipid acyl hydrolase activity which requires serine activesite functionality and at least one additional amino acid residueinteracting with the active site serine is expected to have insectinhibitory bioactivity which can be determined by placing an insectinhibitory amount of the native protein sequence into a bioassay with asusceptible insect to determine insect inhibitory bioactivity. A nativeprotein, mutagenized to inactivate one or more of the residues involvedin active site lipid acyl hydrolase activity can be used in a separatebioassay to confirm the related active site residue involvement ininsect inhibitory bioactivity.

[0021] A further object of the present invention is to providecompositions which protect a plant or a plant part from insectinfestation by one or more of insects selected from the group consistingof corn rootworm, cutworm, wire worm earworm, aphids, piercing andsucking insects, borers, army worms, and potato beetles.

[0022] A further object of the present invention is to provide a methodfor constructing transformed plant cells comprising a DNA sequenceencoding a novel lipid acyl hydrolase having insect inhibitorybioactivity, wherein the hydrolase and insect inhibitory activity areidentified by first treating the hydrolase with diisopropylfluorophosphate to identify at least one serine residue involved inlipid acyl hydrolase activity; second determining the crystal structureof the hydrolase and forming a three dimensional model of the hydrolase;and third, using the three dimensional model of the structure toidentify additional residues involved in active site catalysis; whereinthe transformed plant cells are resistant to insect infestation orinhibit insects upon ingestion of said transformed plant cells. Usingalanine scanning mutagenesis, other residues can be identified which arecrucial for hydrolase enzyme stability. An enzyme displaying lipid acylhydrolase activity which requires serine active site functionality andat least one additional amino acid residue interacting with the activesite serine is expected to have insect inhibitory bioactivity which canbe determined by placing an insect inhibitory amount of cells expressingthe native protein sequence into a bioassay with a susceptible insect todetermine insect inhibitory bioactivity. A native protein, mutagenizedto inactivate one or more of the residues involved in active site lipidacyl hydrolase activity can be used in a separate bioassay to confirmthe related active site residue involvement in insect inhibitorybioactivity.

[0023] Another aspect of the present invention is directed to providingan insect inhibitory composition which prevents or delays thedevelopment of insect resistance to an insect inhibitory compound in afield of crops. The composition contains two or more insect inhibitorycomponents, each component being present in an amount sufficient toinhibit the same insect species, at least one of the components being anovel lipid acyl hydrolase having insect inhibitory bioactivity, whereinthe hydrolase and insect inhibitory activity are identified by firsttreating the hydrolase with diisopropyl fluorophosphate to identify aserine residue involved in lipid acyl hydrolase activity; seconddetermining the crystal structure of the hydrolase and forming a threedimensional model of the hydrolase; and third, using the threedimensional model of the structure to identify additional residuesinvolved in active site catalysis; wherein the composition insectinfestation or inhibit insects upon ingestion of said transformed plantcells.

[0024] An additional aspect of the present invention comprises applyingan insect to inhibitory effective amount of a protein sequencedisplaying lipid acyl hydrolase activity to a plant or incorporatingsaid amount into said plant, wherein said protein sequence displayinglipid acyl hydrolase activity comprises a first peptide sequencecomprising Gly-Xxx₁-Ser-Xxx₂-Gly, and a second peptide sequencecomprising Glu-Xxx₃-Xxx₄-Leu-Val-Asp-Gly. Xxx₁ or Xxx₂ can be threonineor any other amino acid which is structurally and functionally similarto threonine. Xxx₃ can be an aromatic amino acid residue, or preferablytyrosine or phenylalanine. Xxx₄ can be an amino acid residue consideredin the art to be a base, preferably asparagine or histidine. A catalyticactive site structure utilizing a serine-aspartate dyad chemistry issupported by the requirement for both peptide sequences being present,along with three dimensional modeling based on crystal structure of theprotein sequence, and a pH rate profile indicating that a single residuewith a pKa of less than about 5 must be deprotonated to show hydrolaseactivity and insect inhibitory bioactivity.

DESCRIPTION OF THE FIGURES

[0025] The following figures form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0026]FIG. 1 illustrates the cDNA and amino acid sequence translation ofa native patatin isoform designated as Pat17

[0027]FIG. 2 illustrates the hydrolase activity of the alanine scanningvariant isoforms of Pat17

[0028]FIG. 3 illustrates the pH rate profile for the native Pat17 enzyme

[0029]FIG. 4 illustrates the effect of Pat17 and variants on growth ofneonate SCRW larvae

[0030]FIG. 5 illustrates the effect of wild type and H109N variant Pat17on growth of neonate SCRW larvae

[0031]FIG. 6 illustrates (a) a ribbon diagram of the Pat17 structurebased on X-ray crystallography solution analysis; and (b) a ribbondiagram of the proposed Pat17 active site showing the catalytic serineand aspartate residues

[0032]FIG. 7 illustrates a ribbon diagram of the Pat17 structure arounda histidine at position 109

[0033]FIG. 8 illustrates the proposed catalytic mechanism of Pat17involving serine and aspartate catalytic active site residues in acatalytic dyad

[0034]FIG. 9 illustrates the alignment of Pat17 with other dicot patatinor patatin related amino acid sequences, and alignment with severalmonocot patatin related sequences, displaying the conserved catalyticserine motif and catalytic aspartate motif alignments and sequenceconservations.

[0035]FIG. 10 illustrates construction of nucleic acid sequencesencoding patatin permutein proteins, and for illustrative purposes abreakpoint at position 247 is shown.

DESCRIPTION OF THE SEQUENCE LISTINGS

[0036] The following description of the sequence listing forms part ofthe present specification and is included to further demonstrate certainaspects of the present invention. The invention can be better understoodby reference to one or more of these sequences in combination with thedetailed description of specific embodiments presented herein. SEQ IDNO: 1 patatin homolog Pat17 amino acid sequence (Solanum cardiophyllum)SEQ ID NO: 2 patatin isozyme PatFm (mature protein lacking signalpeptide) SEQ ID NO: 3 Patatin isozyme PatIm (mature protein lackingsignal peptide) SEQ ID NO: 4 Patatin isozyme PatL+ (including signalpeptide) SEQ ID NO: 5 Patatin isozyme PatA+ (including signal peptide)SEQ ID NO: 6 Patatin isozyme PatB+ (including signal peptide) SEQ ID NO:7 patatin homolog pentin 1 (Pentaclethra macroloba) SEQ ID NO: 8 monocotpatatin homolog 5c9 (Zea mays) SEQ ID NO: 9 maize patatin homolog aminoacid sequence corn1 SEQ ID NO: 10 maize patatin homolog amino acidsequence corn2 SEQ ID NO: 11 maize patatin homolog amino acid sequencecorn3 SEQ ID NO: 12 maize patatin homolog amino acid sequence corn4 SEQID NO: 13 maize patatin homolog amino acid sequence corn5 SEQ ID NO: 14Serine active site consensus sequence motif SEQ ID NO: 15 Aspartateactive site consensus sequence motif SEQ ID NO: 16 linker sequence SEQID NO: 17 linker sequence SEQ ID NO: 18 oligonucleotide sequence SEQ IDNO: 19 oligonucleotide sequence SEQ ID NO: 20 pMON37402 sequenceencoding permutein protein SEQ ID NO: 21 Permutein protein encoded frompMON37402 sequence SEQ ID NO: 22 pMON37405 sequence encoding permuteinprotein SEQ ID NO: 23 Permutein protein encoded by pMON37405 sequenceSEQ ID NO: 24 pMON37406 sequence encoding permutein protein SEQ ID NO:25 Permutein protein encoded by pMON37406 sequence SEQ ID NO: 26pMON37407 sequence encoding permutein protein SEQ ID NO: 27 Permuteinprotein encoded by pMON37407 sequence SEQ ID NO: 28 pMON37408 sequenceencoding permutein protein SEQ ID NO: 29 Permutein protein encoded bypMON37408 sequence SEQ ID NO: 30 pMON40701 sequence encoding permuteinprotein SEQ ID NO: 31 Permutein protein encoded by pMON40701 sequenceSEQ ID NO: 32 pMON40703 sequence encoding permutein protein SEQ ID NO:33 Permutein protein encoded by pMON40703 sequence SEQ ID NO: 34pMON40705 sequence encoding permutein protein SEQ ID NO: 35 Permuteinprotein encoded by pMON40705 sequence SEQ ID NO: 36 corn homolog peptideSEQ ID NO: 37 patatin homolog Pat17 nucleic acid coding sequence andamino acid translation (Solanum cardiophyllum) SEQ ID NO: 38 DNAsequence encoding a patatin (acyl lipid hydrolase) protein SEQ ID NO: 39potato patatin protein sequence SEQ ID NO: 40 Pre-cleavage patatinprotein produced in Pichia pastoris SEQ ID NO: 41 Post-cleavage patatinprotein produced in Pichia pastoris SEQ ID NO: 42 Conserved Basic aminoacid consensus motif F-Y-X1-E-H/N-G-P SEQ ID NO: 43-60 oligonucleotides

DEFINITIONS

[0037] The following definitions are provided in order to aid thoseskilled in the art in understanding the detailed description of thepresent invention.

[0038] “Chimeric” refers to a fusion nucleic acid or protein sequence. Achimeric nucleic acid sequence is comprised of two sequences joinedin-frame that encode a chimeric protein. The coding regions of multipleprotein subunits may be joined in-frame to form a chimeric nucleic acidsequence that encodes a chimeric protein sequence.

[0039] “Coding sequence”, “open reading frame”, and “structuralsequence” refer to the region of continuous sequential nucleic acidtriplets encoding a protein, polypeptide, or peptide sequence.

[0040] “Codon” refers to a sequence of three nucleotides that specify aparticular amino acid.

[0041] “Complementarity” refers to the specific binding of adenine tothymine (or uracil in RNA) and cytosine to guanine on opposite strandsof DNA or RNA.

[0042] “Deallergenize” (render hypoallergenic) refers to the method ofengineering or modifying a protein such that it has a reduced oreliminated ability to induce an to allergic response. A deallergenizedprotein may be referred to as being hypoallergenic. The degree ofdeallergenization of a protein may be measured in vitro by the reducedbinding of IgE antibodies.

[0043] “DNA sequence heterologous to the promoter region” means that thecoding DNA sequence does not exist in nature in the same gene with thepromoter to which it is now attached.

[0044] “DNA sequence” refers to a DNA molecule that has been isolatedfree of total genomic DNA of a particular species.

[0045] “Electroporation” refers to a method of introducing foreign DNAinto cells that uses a brief, high voltage dc charge to permeabilize thehost cells, causing them to take up extra-chromosomal, epi-genetic DNA,or any nucleotide or polynucleotide molecule provided exogeneously tothe cells.

[0046] “Encoding DNA” refers to chromosomal DNA, plasmid DNA, cDNA, orsynthetic DNA which encodes any of the enzymes or proteins discussedherein.

[0047] “Endogenous” refers to materials originating from within anorganism or cell.

[0048] “Endonuclease” refers to an enzyme that hydrolyzes doublestranded DNA at internal locations.

[0049] “Epitope” refers to a region on an allergen that interacts withthe cells of the immune system. Epitopes are often further defined bythe type of antibody or cell with which they interact, e.g. if theregion reacts with B-cells or antibodies (IgE), it is called a B-cellepitope.

[0050] “Exogenous” refers to materials originating from outside of anorganism or cell. This typically applies to nucleic acid molecules usedin producing transformed or transgenic host cells and plants.

[0051] “Expressibly coupled”, “expressibly linked”, “operably linked”,and “operatively linked”, refer to a promoter or promoter region and acoding or structural sequence in such an orientation and distance thattranscription of the coding or structural sequence may be directed bythe promoter or promoter region. 3′ transcription termination andpolyadenylation sequences can also be operably linked to codingsequences.

[0052] “Expression” refers to the transcription of a gene to produce thecorresponding mRNA and translation of this mRNA to produce thecorresponding gene product, i.e., a peptide, polypeptide, or protein.Expression can also refer to the transcription of a gene coding for atRNA or a structural, catalytic, or functional RNA molecule which is nototherwise subsequently translated into protein.

[0053] “Fusion modified gene” refers to a nucleic acid sequence of oneorigin fused to a nucleic acid sequence from another origin at eitherthe N-termini or the C-termini, e.g. a nucleic acid sequence encoding aninsecticidal protein or fragment from B.t. fused to the N- or C-terminito a nucleic acid sequence encoding patatin or a fragment of patatin orvice versa.

[0054] “Heterologous DNA” refers to DNA from a source different thanthat of the recipient cell.

[0055] “Homologous DNA” refers to DNA from the same source as that ofthe recipient cell.

[0056] “Identity” refers to the degree of similarity between two nucleicacid or protein sequences. An alignment of the two sequences isperformed by a suitable computer program. A widely used and acceptedcomputer program for performing sequence alignments is CLUSTALW v1.6(Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994). The number ofmatching bases or amino acids is divided by the total number of bases oramino acids, and multiplied by 100 to obtain a percent identity. Forexample, if two 580 base pair sequences had 145 matched bases, theywould be 25 percent identical. If the two compared sequences are ofdifferent lengths, the number of matches is divided by the shorter ofthe two lengths. For example, if there were 100 matched amino acidsbetween 200 and a 400 amino acid proteins, they are 50 percent identicalwith respect to the shorter sequence. If the shorter sequence is lessthan 150 bases or 50 amino acids in length, the number of matches aredivided by 150 (for nucleic acid bases) or 50 (for amino acids), andmultiplied by 100 to obtain a percent identity.

[0057] “IgE” (Immunoglobulin E) refers to a specific class ofimmunoglobulin secreted by B cells. IgE binds to specific receptors onMast cells. Interaction of an allergen with mast cell-bound IgE maytrigger allergic symptoms.

[0058] “Immunotherapy” refers to any type of treatment that targets theimmune system. Allergy immunotherapy is a treatment in which aprogressively increasing dose of an allergen is given in order to inducean immune response characterized by tolerance to the antigen/allergen,also known as desensitization.

[0059] “In vitro” refers to in the laboratory.

[0060] “In vivo” refers to in a living organism.

[0061] “Insect inhibitory polypeptide” refers to a polypeptide havingproperties that adversely affects the growth and development of insectpests. Insect inhibitory also refers to isolated nucleic acid moleculescomprising nucleotide sequences encoding polypeptides or proteinsexhibiting insect inhibitory activity, wherein said activity ismanifested by inhibiting the growth or development of, or contributingsubstantially to, or causing the death of a Coleopteran, a Dipteran, aLepidopteran, a Hemipteran, a Hymenopteran, or a sucking and piercinginsect or insect larvae thereof. Insect inhibitory also includesnucleotide sequences encoding novel proteins comprising polypeptideswhich augment the activity of peptides exhibiting insect inhibitoryactivity when fed to Coleopteran, Dipteran, Lepidopteran, Hemipteran,Hymenopteran, or sucking and piercing insects or insect larvae thereof.

[0062] “Monocot” refers to plants having a single cotyledon (the firstleaf of the embryo of seed plants); examples include cereals such asmaize, rice, wheat, oats, and barley.

[0063] “Multiple cloning site” refers to an artificially constructedcollection of restriction enzyme sites in a vector that facilitatesinsertion of foreign DNA into the vector.

[0064] “Mutation” refers to any change or alteration in the sequence ofa gene. Several types exist, including point, frame shift, and splicing.

[0065] “Native” refers to two segments of nucleic acid naturallyoccurring in the same organism. For example, a native promoter is thepromoter naturally found with a given gene in an organism.

[0066] “Naturally occurring” refers to a nucleic acid or protein whichis found in nature, and has not been manipulated or altered by the handof man.

[0067] “Non-naturally occurring” refers to a nucleic acid or proteinwhich is not found in nature, but instead has been synthesized toexhibit properties that are otherwise found in nature. The synthesis ofany such non-naturally occurring nucleic acid or protein does notnecessarily require that the entire sequence of either be syntheticallyproduced, but that only an insubstantial modification, such as anucleotide substitution in a nucleic acid sequence or an amino acidsubstitution in the amino acid sequence of a protein, is all that isnecessary to qualify the nucleic acid or protein as one which isnon-naturally occurring.

[0068] “Nucleic acid segment” or “nucleic acid sequence” is a nucleicacid molecule that has been isolated free of total genomic DNA of aparticular species, or that has been synthesized. Included with the term“nucleic acid segment” are DNA segments or DNA sequences, recombinantvectors, plasmids, cosmids, phagemids, phage, viruses, etcetera.

[0069] “Nucleic acid” refers to deoxyribonucleic acid (DNA) andribonucleic acid (RNA).

[0070] Nucleic acid codes: A=adenosine; C=cytosine; G=guanosine;T=thymidine; N=equimolar A, C, G, and T; I=deoxyinosine; K=equimolar Gand T; R=equimolar A and G; S=equimolar C and G; W=equimolar A and T;Y=equimolar C and T.

[0071] “Open reading frame (ORF)” refers to a region of DNA or RNAencoding a peptide, polypeptide, or protein.

[0072] “Plasmid” refers to a circular, extrachromosomal,self-replicating DNA.

[0073] “Point mutation” refers to an alteration of a single nucleotidein a nucleic acid sequence.

[0074] “Polymerase chain reaction (PCR)” or thermal amplification refersto an enzymatic technique to create multiple copies of one sequence ofnucleic acid. Copies of DNA sequence are prepared by shuttling a DNApolymerase between two amplimers. The basis of this amplification methodis multiple cycles of temperature changes to denature, then re-annealamplimers, followed by extension to synthesize new DNA strands in theregion located between the flanking amplimers.

[0075] “Probe” refers to a polynucleotide sequence which iscomplementary to a target polynucleotide sequence in the analyte.

[0076] “Promoter” or “promoter region” refers to a DNA sequence, usuallyfound upstream of, or positioned 5′ with reference to, a codingsequence, that controls expression of the coding sequence by controllingproduction of messenger RNA (mRNA) by providing the recognition site forRNA polymerase and/or other factors necessary for transcriptioninitiation at the correct site. As contemplated herein, a promoter orpromoter region includes variations of promoters derived by means ofligation to various regulatory sequences, random or controlledmutagenesis, and addition or duplication of enhancer sequences. Thepromoter regions disclosed herein, and biologically functionalequivalents thereof, are responsible for driving the transcription ofcoding sequences under their control when introduced into a host as partof a suitable recombinant vector, as demonstrated by its ability toproduce mRNA.

[0077] “Recombinant DNA construct” or “recombinant vector” refers to anyagent such as a plasmid, cosmid, virus, autonomously replicatingsequence, phage, or linear or circular single-stranded ordouble-stranded DNA or RNA nucleotide sequence, derived from any source,capable of genomic integration or autonomous replication, comprising aDNA molecule in which one or more DNA sequences have been linked in afunctionally operative manner. Such recombinant DNA constructs orvectors are capable of introducing a 5′ regulatory sequence or promoterregion and a DNA sequence for a selected gene product into a cell insuch a manner that the DNA sequence is transcribed into a functionalmRNA which is translated and therefore expressed. Recombinant DNAconstructs or recombinant vectors may be constructed to be capable ofexpressing antisense RNA's, in order to inhibit translation of aspecific RNA of interest.

[0078] “Recombinant proteins”, also referred to as “heterologousproteins”, are proteins which are normally not produced by the hostcell.

[0079] “Regeneration” refers to the process of growing a plant from aplant cell (e.g., plant protoplast or explant).

[0080] “Regulatory sequence” refers to a nucleotide sequence locatedupstream (5′), within, and/or downstream (3′) to a DNA sequence encodinga selected gene product whose transcription and expression is controlledby the regulatory sequence in conjunction with the protein syntheticapparatus of the cell.

[0081] “Restriction enzyme” refers to an enzyme that recognizes aspecific palindromic sequence of nucleotides in double stranded DNA andcleaves both strands; also called a restriction endonuclease. Cleavagetypically occurs within the restriction site.

[0082] “Result-effective substitution” (RES) refers to an amino acidsubstitution within an IgE-binding region (epitope) of a protein(patatin) which reduces or eliminates the IgE binding by that epitope.

[0083] “Selectable marker” refers to a nucleic acid sequence whoseexpression confers a phenotype facilitating identification of cellscontaining the nucleic acid sequence. Selectable markers include thosewhich confer resistance to toxic chemicals (e.g. ampicillin resistance,kanamycin resistance), complement a nutritional deficiency (e.g. aninability to produce any or produce sufficient compounds for survivalwithout supplementation such as uracil, histidine, leucine,diaminopimelic acid, etc.), or impart a visually or opticallydistinguishing characteristic (e.g. color changes or fluorescence).

[0084] “Transcription” refers to the process of producing an RNA copyfrom a DNA template. Reverse transcription refers to the process ofproducing either an RNA copy from an RNA template, or a DNA copy from anRNA template.

[0085] “Transformation” refers to a process of introducing an exogenousnucleic acid sequence (e.g., a vector, recombinant nucleic acidmolecule) into a cell or protoplast in which that exogenous nucleic acidis incorporated into a chromosome or into a naturally occurringheterologous DNA, such as into chloroplast DNA, or is capable ofautonomous replication.

[0086] “Transformed cell” is a cell whose DNA has been altered by theintroduction of an exogenous nucleic acid molecule into that cell.

[0087] “Transgenic cell” refers to any cell derived from or regeneratedfrom a transformed cell or derived from a transgenic cell. Exemplarytransgenic cells include plant calli derived from a transformed plantcell and particular cells such as leaf, root, stem, e.g., somatic cells,or reproductive (germ) cells obtained from a transgenic plant.

[0088] “Transgenic plant” refers to a plant or progeny thereof derivedfrom a transformed plant cell or protoplast, wherein the plant DNAcontains an introduced exogenous nucleic acid sequence not originallypresent in a native, non-transgenic plant of the same species.Alternatively, the plant DNA may contain the introduced nucleic acidsequence in a higher copy number than in the native, non-transgenicplant of the same species.

[0089] “Translation” refers to the production of protein from messengerRNA.

[0090] “Vector” refers to a plasmid, cosmid, bacteriophage, or virusthat carries foreign DNA into a host organism.

[0091] “Western blot” refers to protein or proteins that have beenseparated by electrophoresis, transferred and immobilized onto a solidsupport, then probed with an antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0092] The present invention is directed to the art areas of plantmolecular biology, plant agriculture, and entomology as well as toprotein chemistry, immunology, and protein crystallography.

[0093] Economically important crops have always been subject to insectinfestation, at times resulting in devastating damage. Even when damageis not ultimately devastating, the insect pressure can significantlyalter the yield and quality of the harvest. Means for controlling theinsect pressure in a field of crops has been partially addressed bychemical applications as well as, to a lesser extent, traditionalbreeding methodologies. True to genetic variability, however, theinsects seem to adapt readily to these traditional means for control.Naturally occurring plant traits which confer insect inhibitoryadvantages have evolved and been selected for by plant breeders overgenerations of breeding. These traits have either succumbed to, or arelikely to ultimately succumb to races of insects which adapt to feedseemingly unaffected by the selected traits. Although such naturallyselected plant derived traits are in fact useful, they are notaltogether the most effective means of combating insect pressure for anumber of reasons. First, the tolerances that plants can evolve are inconstant flux with the changes that insects accrue in order to overcomethe defenses. Second, and perhaps more importantly, the rate at whichtraditional breeding takes place is too slow and cumbersome to providethe types of resistance that are necessary to maintain the defenses forcrop plants. In addition, other means have proven much more effective inconferring insect pressure control.

[0094] One such means is topical chemical treatment to susceptibleplants. This has particular advantages because it can be applied onlywhen insect pressure is detected, and only in amounts necessary toattempt to achieve control of the insect pressures. However, there aresubstantial disadvantages to chemical treatments. Primarily, mostchemical applications utilize organophosphates or similar compositionswhich are not only toxic to the target insect pests but to all otherinsect, arachnid, mammalian or avian species present in the localenvironment to which the application is directed. Second, application ofindividual chemical compositions leads to rapid development ofresistance to the composition. There has been good success in treatingfields of crops, however, with compositions containing two or morechemical insecticides, at least one of which acts to inhibit, kill, orotherwise control at least the target insect pest using a mode of actiondifferent from the other pesticides present in the composition. Thismeans also leads to virtually no development of resistance. A thirddisadvantage to using chemical treatments is that often the compositionis wholly or partially non-biodegradable and therefore not abio-efficacious means for treating crops in a field in which further useof the field for crop rotations is contemplated. In addition, anotherdisadvantage to topical applications is that many insect pests areshielded from the topical effects of the treatments because of thenature of their life cycles. Insects such as grubs, borers, and leafrollers con continue to feast uninhibited because of the nature of theirchosen ecological niche. Therefore, alternative means of controllinginsect pressures have been necessary.

[0095] Through the advent of molecular biology, recombinant plantsexpressing very effective insect control proteins have developed andrecently deployed into commercial varieties which can now be obtainedthrough seed providers. Such recombinant plants generally contain geneswhich have been manipulated to enable the plants to express proteinseither identical to or substantially identical to naturally occurringproteins isolated from Bacillus thuringiensis species of bacteria. Suchproteins, designated through nomenclature as insecticidal crystalproteins or ICP's or BT's, have been very effective in most plants whichhave been genetically altered to express them. However, these proteinsare also susceptible to the development of resistance in various targetinsects. For example, the Cry3 class of proteins are BT ICP's which areparticularly effective in controlling, inhibiting, or killing variousColeopteran species of insect larvae. Some members of this particularclass are now used preferentially to control corn rootworms. However, itis presumed that when expressed alone in plants without some additionalcoleopteran effective treatment, a coleopteran larvae feeding on such aplant would eventually develop some level of resistance to the BT ICP,diminishing the effectiveness of the recombinant trait in the crop, andrendering valueless the efforts in procuring such recombinant varieties.The application of an additional treatment in combination with the BTICP that had a separate mode of action when compared to the BT ICP andwhich was equally effective in controlling coleopteran species woulddiminish to vanishingly small the likelihood that resistant races of thetarget coleopteran species would develop at all One report has suggestedthat the co-expression of two or more BT ICP's in a plant, wherein eachBT ICP was toxic to the same insect species but wherein each BT ICPexpressed did not bind competitively to insect brush border membranevesicle receptors, would diminish the likelihood that insect resistancewould develop to any of the BT ICP's present in that plant. (VanMellaert et al. U.S. Pat. No. 5,866,784; Feb. 2, 1999). However,although there are a variety of classes of BT ICP proteins, with eachclass of protein being particularly effective in controlling a class ofinsect species, such as Cry1's effectiveness vs lepidopterans, Cry2'seffectiveness in controlling some lepidopterans but many which also haveeffects on dipterans, and Cry3's effectiveness in controlling someColeopteran's, there are only a limited number of Cry proteins whichcould be used in the manner described. This lack of numerosity andvariety is particularly true for the Cry3 class of proteins, ie thosewhich are preferentially effective in controlling various Coleopteranspecies. In addition, more sensitive methods for measuring binding of BTICP's to insect brush border membrane vesicle receptors have beendeveloped since the methods as taught in Van Mellaert et al. The moresensitive methods suggest that even for those pairs of BT ICP's whichVan Mellaert et al. demonstrated non-competitive binding, there appearsin fact to be some competitive binding taking place, making it morelikely that when two or more BT ICP's are used in combination which donot completely exhibit non-competitive binding, resistance to both BTICP's could develop more rapidly than previously believed. Therefore,there is a need to identify and/or develop additional insect inhibitoryproteins which do not act in the same way, ie using the same mode ofaction, as BT ICP's.

[0096] A variety of plant, bacterial, and fungal derived proteins havebeen identified which display insect inhibitory activity. Some of theseinclude plant lectins, and as described above, other insect inhibitoryproteins derived from Xenorhabdus and/or Photorhabdus species ofbacteria. It is not clear whether these proteins act in modes differentfrom that of the BT ICP's. It is clear, however, that there isincreasing disinterest by various groups in having plants which expressforeign proteins, ie proteins that are not otherwise naturally occurringin plants. It may be more acceptable to such groups to engineer plantswhich express useful proteins which have been derived from heterologousplant sources, or more preferably from homologous plant sources. Inparticular, identification of plant proteins which have properties ofinsect inhibition or insect control when ingested by insect pests, andwhich function in a way which is different from the function of BT ICP'sor other bacterial or heterologous proteins would be particularlyuseful.

[0097] Plant non-specific lipid acyl hydrolases have been identifiedfrom a variety of plant sources including potato tubers, flowers, andleaves, bean leaves and rice bran as well as many other plant sources.The activity of plant non-specific lipid acyl hydrolases is extremelyhigh in many tissues, and although their action in causing rancidity instored agricultural products and in damaged or infected tissues has beenwell documented, their in vivo physiological role is still uncertain.

[0098] Patatin is a major potato tuber protein that has been shown tohave esterase, lipase, and insect inhibitory activities. This protein isalso classified as a non-specific lipid acyl hydrolase. As used herein,plant non-specific lipid acyl hydrolase includes a protein or proteinsequence having substantial homology to potato patatin based onalignment algorithms and which can be demonstrated to hydrolyze acylgroups from at least one of several classes of lipids, includingglycolipids, phospholipids, sulfolipids, and mono- and di-acylglycerols, but is inactive on triacylglycerols. The acyl hydrolasereleases both fatty acids from diacyl glycerolipids, and in many cases,there is no preference for either the 1- or 2-position of the acyl esterlinkage. Thus, the enzyme possesses a combined catalytic capacity ofphospholipase A1, A2, and B, as well as glycolipase, sulfolipases andmonoacylglycerol lipase. Similarities of the plant non-specific lipidacyl hydrolase enzymes from various tissues include the following: (1)they exert a similar pattern of substrate specificity as describedabove; (2) they may occur as isozymes in each tissue and they havefairly similar patterns of substrate specificity; (3) the activity ratioof the enzyme preparation on galactolipid and phospholipid remainsfairly constant throughout an enzyme purification procedure; and (4) theenzyme carries out acyltransferase reactions with each of the substrates(Gailliard, in “The Biochemistry of Plants”, P. K. Stumpf and E. E.Conn, eds., v4:85-116, Academic Press, New York, 1980).

[0099] The best characterized plant non-specific lipid acyl hydrolase ispatatin, isolated from potato tubers. Patatin is a mixture of at least 6to 10 closely related polypeptides, isoforms, or isozymes which differin their primary amino acid sequence, patterns of glycosylation, andhydrolytic activities (Hofgren et al., Plant Sci. 66:221-230, 1990).These proteins are encoded by a family of about 15 genes per haploidgenome, and genes encoding several patatin isoforms have been sequencedand published (Mignery et al., Nucl. Acids Res. 12:7987-8000, 1984).Sequences encoding additional patatin related proteins from potato andfrom corn are set forth herein.

[0100] Patatin is synthesized as an approximately 43,000 Dalton (43 kDa)preprotein with a short signal peptide for targeted secretion into theER and subsequent passage through the Golgi apparatus. The signalpeptide is cleaved upon insertion of the mature peptide into the lumenof the ER and the mature form of patatin is glycosylated in the Golgi tobecome a mature protein of about 40 kDa. One skilled in the art willrecognize that variant patatins or patatin related sequences displayingnon-specific lipid acyl hydrolase activity and insect inhibitorybioactivity can vary by as much as 10-15 percent in size from the majorpotato patatin sequence. In any event, the present inventionspecifically contemplates the use of any of the patatin isoforms. It hasbeen identified as a part of the inventions described herein thatvariations may exist in the amino acid sequence of patatin and relatedproteins without any significant effect on its functionalcharacteristics. However, any changes to active site amino acid sequencemotifs as disclosed herein have substantial impact on the enzymatic andinsect inhibitory bioactivity, and therefore should be avoided whenconstruing patatin homologs for use as contemplated herein.

[0101] Biochemical assays which monitor the lipolytic or esterolyticactivity of plant non-specific lipid acyl hydrolases are useful forensuring that proteins isolated from plant tissues are in fact lipidacyl hydrolases. To ensure that the enzyme activity observed in suchassays is due to protein activity, protease sensitivity can be-measured. In addition, insect bioassays are useful as monitors for theinsect inhibitory activity displayed by non-specific lipid acylhydrolases. One skilled in the art would know how to backtranslate froman amino acid sequence to obtain a DNA sequence which could besynthesized as a redundant probe to identify one or more genomic or cDNAsequences encoding one or more plant non-specific lipid acyl hydrolases.In fact, using the active site amino acid sequence motifs disclosedherein, one skilled in the art could easily identify any plantnon-specific lipid acyl hydrolase from any plant tissue, whether monocotor dicot species.

[0102] Based on the analysis of the amino acid sequence of patatin, ithas been previously shown that a serine residue is required for lipidacyl hydrolase activity as well as for insect inhibitory bioactivity,and that the serine residue within the amino acid sequence motifGly-Xxx₁-Ser-Xxx₂-Gly (SEQ ID NO:14) is the catalytic serine residue.This disclosure reports the isolation of a single potato patatinisozyme, designated Pat17, and reports the results of alanine scanningmutagenesis of the gene encoding the protein to identify the likelycatalytic residues responsible for both the esterase and insectinhibitory bioactivity. In addition, the active site amino acid sequencemotif containing a required serine residue was altered to assess itsrole in catalytic function. A set of 75 amino acid sequence variantswere generated using site-directed mutagenesis, expressed in the yeastPichia pastoris, and analyzed for esterase activity. The variantsidentified using alanine scanning mutagenesis and displaying lowesterase activity were purified and assayed for insect inhibitoryactivity. The inventors have herein identified Ser77 and Asp215 residuesin Pat17 to be critical for both esterase and insect inhibitorybioactivity. The substitution of Ser77 with cysteine, alanine,aspartate, threonine, or asparagine residues significantly reduced boththe esterase and insect inhibitory activity, further supporting the roleof Ser77 in maintaining the activity of the protein. The pH rate profileof the protein indicates that a single residue with a pKa of less thanabout 5 must be deprotonated for the protein to show activity, whichsupports the role of Asp215 as a catalytic residue. Surprisingly,substitution of three His residues with alanine in Pat17 did not producean inactive enzyme. His variant H109A could not be expressed. Anisosteric change at this position, H109N, maintained full esterase andbioactivity. Other amino acid variations at position 109 includedcysteine, aspartate, and arginine. These variants were also unable to beexpressed, suggesting that His109 does not play a direct role incatalysis but instead is implicated as important in the stability of theprotein, as suggested by the X-ray crystal structure. The X-Ray crystalstructure solution, reported herein, along with the alanine scanningmutagenesis and the amino acid sequence alignments with other sequenceshaving substantial homology to potato patatin further supports therequirement for serine and aspartate in catalysis and insect inhibitionand further provides a means for identifying any member of a family ofconserved plant proteins displaying non-specific lipid acyl hydrolaseactivity and insect inhibitory bioactivity and which utilizing serineand aspartate in maintaining these functions (FIG. 9). In particular thealignments have allowed the identification of consensus sequences which,when coupled with X-Ray crystallographic data on at least one of thealigned protein sequences, allows the identification of the residueswhich fold into the active site of the enzyme and which are necessaryfor maintaining lipid acyl hydrolase activity and insect inhibitorybioactivity. These alignment consensus sequences are set forth in FIG. 9as underlined sequences and in SEQ ID NO:14 (Gly-Xaa₁-Ser-Xaa₂-Gly) andSEQ ID NO:15 (Glu-Xaa₁-Xaa₂-Leu-Val-Asp-Gly). Xaa₁ and Xaa₂ as set forthin SEQ ID NO:14 can be either Ser or Thr. Xaa₁ as set forth in SEQ IDNO:15 can be any of the aromatic amino acids such as Tyr, Phe, Trp, andpreferably are either Tyr or Phe. Xaa₂ as set forth in SEQ ID NO:15 canbe generally be a basically charged amino acid such as His or Asn, witha preference for either being equally weighted.

[0103] Variants or analogues of patatin or patatin homologs are alsospecifically contemplated herein. Other than the contemplated amino acidsequence variants or variants of varying lengths relative to potatopatatin, each having or retaining acyl hydrolase activity and insectinhibitory bioactivity, other contemplated variants include permuteins.Permuteins are generally proteins that comprise an amino acid sequencenot found in nature, but which, upon three dimensional analysis ormodeling appear to fold in three dimensional space into theconfiguration of the native protein and continue to display at least thesame enzymatic and insect inhibitory bioactivity as the native protein.In addition, it is preferable that the DNA sequence encoding thepermutein display at least the same level of expression in host cells asa codon optimized DNA sequence encoding the native protein sequence.Herein, once the crystal structure of a protein is solved, if thecarboxy and amino termini of the protein are near enough to one another,ie within about 50 Å, then one or more breakpoints within the proteinsequence structure can be selected so that the ends of the breakpoint(s)form the new amino and carboxy termini of the resultant protein, thepermutein which is then joined into a single contiguous amino acidsequence by constructing a DNA sequence encoding the new, novel proteinsequence such that the old carboxy terminus codon is adjacent to andupstream of the original native amino terminal amino acid codon.

[0104] The positions of the internal breakpoints described herein arefound on the protein surface, and are distributed throughout the linearsequence without any obvious bias towards the ends or the middle.Breakpoints occurring below the protein surface may additionally beselected. The rearranged two subunits may be joined by a peptide linker.A preferred embodiment involves the linking of the N-terminal andC-terminal subunits by a three amino acid linker, although linkers ofvarious sizes may be used. Additionally, the N-terminal and C-terminalsubunits may be joined lacking a linker sequence. Furthermore, a portionof the C-terminal subunit may be deleted and the connection made fromthe truncated C-terminal subunit to the original N-terminal subunit andvice versa as previously described (Yang and Schachman, Proc. Natl.Acad. Sci. U.S.A., 90: 11980-11984, 1993; Viguera, et al., Mol. Biol.,247: 670-681, 1995; Protasova, et al., Prot. Eng., 7:1373-1377, 1994).

[0105] The novel insecticidal proteins of the present invention may berepresented by the formula:

X¹-(L)_(a)-X²

[0106] wherein;

[0107] a is 0 or 1, if a is 0, then the permutein does not contain alinker sequence;

[0108] X¹ is a polypeptide sequence corresponding to amino acids n+1through J;

[0109] X² is a polypeptide corresponding to amino acids 1 through n;

[0110] n is an integer ranging from 1 to J−1;

[0111] J is an integer greater than n+1; and

[0112] L is a linker.

[0113] In the formula above, the constituent amino acid residues of thenovel insect inhibitory protein are numbered sequentially 1 through Jfrom the original amino terminus to the original carboxyl terminus. Apair of adjacent amino acids within this protein may be numbered n andn+1 respectively where n is an integer ranging from 1 to J−1. Theresidue n+1 becomes the new N-terminus of the novel insect inhibitoryprotein and the residue n becomes the new C-terminus of the novel insectinhibitory protein.

[0114] For example, a parent protein sequence consisting of 120 aminoacids may be selected as a starting point for designing a permutein(J=120). If the breakpoint is selected as being between position 40 andposition 41, then n=40. If a linker is selected to join the twosubunits, the resulting permutein will have the formula: (amino acids41-120)-L-(amino acids 140). If a linker was not used, the resultingpermutein will have the formula: (amino acids 41-120)-(amino acids 140).

[0115] The length of the amino acid sequence of the linker may beselected empirically, by using structural information, or by using acombination of the two approaches. When no structural information isavailable, a small series of linkers may be made whose length can span arange of 0 to 50 Å and whose sequence is chosen in order to besubstantially consistent with surface exposure (Hopp and Woods, Mol.Immunol., 20: 483-489, 1983; Kyte and Doolittle, J. Mol. Biol., 157:105-132, 1982; Lee and Richards, J. Mol. Biol., 55: 379-400, 1971) andthe ability to adopt a conformation which does not significantly affectthe overall configuration of the protein (Karplus and Schulz,Naturwissenschaften, 72: 212-213, 1985). Assuming an average length of2.0 to 3.8 Å per residue, this would mean the length to test would bebetween about 0 to about 30 residues, with 0 to about 15 residues beingthe preferred range. Accordingly, there are many such sequences thatvary in length or composition that can serve as linkers with the primaryconsideration being that they be neither excessively long norexcessively short (Sandhu, et al., Critical Rev. Biotech., 12: 437-467,1992). If the linker is too long, entropy effects may destabilize thethree-dimensional fold and may affect protein folding. If the linker istoo short, it may destabilize the molecule due to torsional or stericstrain.

[0116] Use of the distance between the chain ends, defined as thedistance between the C-alpha carbons, may be used to define the lengthof the sequence to be used, or at least to limit the number ofpossibilities that may be tested in an empirical selection of linkers.Using the calculated length as a guide, linkers with a range of numberof residues (calculated using 2 to 3.8 Å per residue) may be selected.These linkers may be composed of the original sequence, shortened orlengthened as necessary, and when lengthened the additional residues maybe chosen to be flexible and hydrophilic as described above; oroptionally the original sequence may be substituted for using a seriesof linkers, one example being Gly-Pro-Gly (SEQ ID NO:16); or optionallya combination of the original sequence and new sequence having theappropriate total length may be used. An alternative short, flexiblelinker sequence is Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:17).

[0117] Sequences of novel patatin analogs capable of folding tobiologically active molecules may be prepared by appropriate selectionof the beginning (amino terminus) and ending (carboxyl terminus)positions from within the original to polypeptide chain while optionallyusing a linker sequence as described above.

[0118] Amino and carboxyl termini may be selected from within a commonstretch of sequence, referred to as a breakpoint region, using theguidelines described below. A novel amino acid sequence is thusgenerated by selecting amino and carboxyl termini from within the samebreakpoint region. In many cases, the selection of the new termini willbe such that the original position of the carboxyl terminus immediatelypreceded that of the amino terminus. However, selections of terminianywhere within the region may result in a functional protein, and thatthese will effectively lead to either deletions or additions to theamino or carboxyl portions of the new sequence.

[0119] The primary amino acid sequence of a protein dictates folding tothe three-dimensional structure beneficial for expression of itsbiological function. It is possible to obtain and interpretthree-dimensional structural information using X-ray diffraction ofsingle protein crystals or nuclear magnetic resonance spectroscopy ofprotein solutions. Examples of structural information that are relevantto the identification of breakpoint regions include the location andtype of protein secondary structure (alpha and 3-10 helices, paralleland anti-parallel beta sheets, chain reversals and turns, and loops(Kabsch and Sander, Biopolymers, 22: 2577-2637, 1983), the degree ofsolvent exposure of amino acid residues, the extent and type ofinteractions of residues with one another (Chothia, C., Ann. Rev.Biochem., 53: 537-572, 1984), and the static and dynamic distribution ofconformations along the polypeptide chain (Alber and Mathews, MethodsEnzymol., 154: 511-533, 1987). In some cases additional information isknown about solvent exposure of residues, one example is a site ofpost-translational attachment of carbohydrate which is necessarily onthe surface of the protein. When experimental structural information isnot available, or when it is not feasible to obtain the information,methods are available to analyze the primary amino acid sequence inorder to make predictions of protein secondary and tertiary structure,solvent accessibility and the occurrence of turns and loops (Fasman, G.,Ed. Plenum, New York, 1989; Robson, B. and Gamier, J. Nature, 361: 506,1993).

[0120] Biochemical methods may be applicable for empirically determiningsurface exposure when direct structural methods are not feasible; forexample, using the identification of sites of chain scission followinglimited proteolysis in order to infer surface exposure (Gentile, F. andSalvatore, G., Eur. J. Biochem., 218: 603-621, 1993). Thus, using eitherthe experimentally derived structural information or to predictivemethods (Srinivasan, R. and Rose, G. D. Proteins, 22: 81-99, 1995), theparental amino acid sequence may be analyzed to classify regionsaccording to whether or not they are integral to the maintenance ofsecondary and tertiary structure. The sequences within regions that areknown to be involved in periodic secondary structure (alpha and 3-10helices, parallel and anti-parallel beta sheets) are regions that shouldbe avoided. Similarly, regions of amino acid sequence that are observedor predicted to have a low degree of solvent exposure are more likely tobe part of the so-called hydrophobic core of the protein and should alsobe avoided for selection of amino and carboxyl termini. Regions that areknown or predicted to be in surface turns or loops, and especially thoseregions that are known not to be required for biological activity, maybe preferred sites for new amino and carboxyl termini. Stretches ofamino acid sequence that are preferred based on the above criteria maybe selected as breakpoint regions.

[0121] An embodiment of the invention is directed towards patatinpermutein proteins. The permutein proteins preferably maintain esteraseactivity and insect inhibitory properties. The permutein proteinspreferably are less allergenic than the wild type patatin protein toindividuals or animals allergic to potatoes. This may be assayed by thebinding of antibodies to the wild type patatin and patatin permuteinproteins.

[0122] The permutein proteins may optionally contain a linker sequence.The linker may generally be any amino acid sequence, preferably isGly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:17) or Gly-Pro-Gly (SEQ IDNO:16), and more preferably is Gly-Pro-Gly.

[0123] Embodiments of the invention also include isolated nucleic acidmolecule segments comprising a structural nucleic acid sequence encodinga patatin permutein protein. The linker may generally be any amino acidsequence, preferably is Gly-Gly-Gly-Ser-Gly-Gly-Gly or Gly-Pro-Gly, andmore preferably is Gly-Pro-Gly. Alternatively, the encoded patatinpermutein protein may lack a linker sequence.

[0124] An embodiment of the invention is directed towards recombinantvectors which encode a patatin permutein protein. Alternatively, theencoded patatin permutein protein may lack a linker sequence.

[0125] Another preferred embodiment of the present invention encompassescells transformed with the DNA constructs disclosed herein, and by useof the transformation vectors well known in the art. Transformed cellscontemplated in the present invention include both prokaryotic andeukaryotic cells which express the proteins encoded for by the novel DNAconstructs of the present invention. The process of producing transgeniccells is well-known in the art. In general, the method comprisestransforming a suitable host cell with a DNA sequence which contains apromoter operatively linked to a coding region that encodes anon-specific lipid acyl hydrolase. Such a coding region is generallyoperatively linked to a transcription-terminating region, whereby thepromoter is capable of driving the transcription of the coding region inthe cell, and hence providing the cell the ability to produce the enzymein vivo. Alternatively, in instances where it is desirable to control,regulate, or decrease the amount of a particular hydrolase or hydrolasesexpressed in a particular transgenic cell, the invention also providesfor the expression of hydrolase antisense mRNA; intron antisense mRNA;chloroplast targeting antisense mRNA; or five prime untranslated region(UTR) antisense mRNA. The use of antisense mRNA as a means ofcontrolling or decreasing the amount of a given protein of interest in acell is well-known in the art.

[0126] In a preferred embodiment, the invention encompasses a plant cellwhich has been transformed with a nucleic acid sequence or DNA constructof the invention, and which expresses a gene or gene segment encodingone or more of the coleopteran-active non-specific lipid acyl hydrolasesas disclosed herein. As used herein, the term “transgenic plant cell” isintended to refer to a plant cell that has incorporated DNA sequences,including but not limited to genes which are perhaps not normallypresent, DNA sequences not normally transcribed into RNA or translatedinto a protein (“expressed”), or any other genes or DNA sequences whichone desires to introduce into the non-transformed plant, such as geneswhich may normally be present in the non-transformed plant but which onedesires to either genetically engineer or to have altered expression.

[0127] It is contemplated that in some instances the genome of atransgenic plant of the present invention will have been augmentedthrough the stable introduction of a coleopteran active non-specificlipid acyl hydrolase-encoding DNA constructs as disclosed herein. Insome instances, more than one transgene will be incorporated into thenuclear genome, or into the chloroplast or plastid genome of thetransformed host plant cell. Such is the case when more than onehydrolase protein-encoding DNA sequence is incorporated into the genomeof such a plant. In certain situations, it may be desirable to have one,two, three, four, or even more non-specific lipid acyl hydrolaseprotein-encoding polynucleotides (either native orrecombinantly-engineered) incorporated and stably expressed in thetransformed transgenic plant.

[0128] In preferred embodiments, the introduction of the transgene intothe genome of the plant cell results in a stable integration wherein theoffspring of such plants also contain a copy of the transgene in theirgenome. The heritability of this genetic element by the progeny of theplant into which the gene was originally introduced is a preferredaspect of this invention. A preferred gene which may be introducedincludes, for example a plant non-specific lipid acyl hydrolase enzyme,and particularly one or more of those described herein.

[0129] Means for transforming a plant cell and the preparation of atransgenic cell line are well-known in the art (as exemplified in U.S.Pat. Nos. 5,550,318; 5,508,468; 5,482,852; 5,384,253; 5,276,269; and5,225,341, all specifically incorporated herein by reference in theirentirety), and are briefly discussed herein. Vectors, plasmids, cosmids,YACs (yeast artificial chromosomes) and DNA segments for use intransforming such cells will, of course, generally comprise either theoperons, genes, or gene-derived sequences of the present invention,either native, or synthetically-derived, and particularly those encodingthe disclosed crystal proteins. These DNA constructs can further includestructures such as promoters, enhancers, polylinkers, or even genesequences which have positively- or negatively-regulating activity uponthe particular genes of interest as desired. The DNA segment or gene mayencode either a native or modified hydrolase protein, which will beexpressed in the resultant recombinant cells, and/or which will impartan improved phenotype to the regenerated plant.

[0130] Transgenic cells specifically contemplated in the presentinvention include transgenic plant cells. Particularly preferred plantcells include those cells obtained from corn, wheat, soybean, turfgrasses, ornamental plant, fruit tree, shrubs, vegetables, grains,legumes, and the like, or any plant into which introduction of acoleopteran active non-specific lipid acyl hydrolase transgene isdesired.

[0131] In another aspect, plants transformed with any DNA construct ofthe present invention that express the proteins for which the constructencodes, are contemplated as being a part of this invention.Accordingly, the invention further provides transgenic plants which havebeen transformed with a DNA construct, as disclosed herein, andtransformed by use of transformation vectors as disclosed herein.Agronomic, horticultural, ornamental, and other economically orcommercially useful plants can be made in accordance with the methodsdescribed herein, to express plant non-specific lipid acyl hydrolases atlevels high enough to confer resistance to insect pathogens whileremaining morphologically normal.

[0132] Such plants may co-express the plant non-specific lipid acylhydrolase polypeptide along with other antifungal, antibacterial, orantiviral pathogenesis-related peptides, polypeptides, or proteins;insect inhibitory proteins; proteins conferring herbicide resistance;and proteins involved in improving the quality or quantity of plantproducts or agronomic performance of plants. Simultaneous co-expressionof multiple proteins in plants is advantageous in that it exploits morethan one mode of action to control plant pathogenic damage. This canminimize the possibility of developing resistant pathogen strains,broaden the scope of resistance, and potentially result in a synergisticinsect inhibitory effect, thereby enhancing a plant's ability to resistinsect infestation (Intl. Patent Appl. Publ. No. WO 92/17591, 15 October1992, specifically incorporated herein by reference in its entirety).

[0133] The transformed plant of the current invention may be either amonocotyledonous plant or a dicotyledonous plant. Where the plant is amonocotyledonous plant, it may be any one of a variety of species.Preferred monocotyledonous species encompassed by the present inventionmay include maize, rice, wheat, barley, oats, rye, millet, sorghum,sugarcane, asparagus, turfgrass, or any of a number of other grains orcereal plants. In preferred embodiments, the monocot is a maize plant.

[0134] The present invention also contemplates a variety ofdicotyledonous plants such as cotton, soybean, tomato, potato, citrus,tobacco, sugar beet, alfalfa, fava bean, pea, bean, apple, cherry, pear,strawberry, raspberry, or any other legume, tuber, or fruit plant. Inpreferred embodiments, the dicot is a soybean plant, a tobacco plant, ora cotton plant.

[0135] Many of the plants intended to be transformed according to thedisclosed invention are commercial crop plants. The commercial form ofthese plants may be the original plants, or their offspring which haveinherited desired transgenes. Accordingly, plants further contemplatedwithin the ambit of the present invention include any offspring ofplants transformed with any of the permutations of the DNA constructwhich are noted in this application. Specifically, the offspring may bedefined as an R₀ transgenic plant. Other progeny of the transformedplant are also included within the scope of the present invention,including any progeny plant of any generation of the transformed plant,wherein the progeny plant has inherited the DNA construct from any R₀plant.

[0136] Upon transformation with a specific DNA construct, the nucleicacid or polynucleotide segments of the construct may be incorporated invarious portions into a chromosome of the transformant. Therefore, inanother embodiment, the present invention encompasses any transgenicplant or plant cell prepared by the use of a DNA construct disclosedherein. Such a plant or cell encompassed by the present inventionincludes those prepared by a process which has the following steps: (1)obtaining a DNA construct including a coleopteran active plantnon-specific lipid acyl hydrolase coding region positioned in frame andunder the control of a promoter operable in the plant, and a signalpeptide sequence coding region for ER targeting of the hydrolasepositioned upstream of the plant non-specific lipid acyl hydrolasecoding region and downstream of the promoter; and (2) transforming theplant with the obtained DNA construct, so that the plant expresses theplant non-specific lipid acyl hydrolase. The plant may also have beentransformed so that it further incorporates into its genome andexpresses other insect inhibitory proteins.

[0137] In a related aspect, the present invention also encompasses aseed produced by the transformed plant, a progeny from such seed, and aseed produced by the progeny of the original transgenic plant, producedin accordance with the above process. Such progeny and seeds will have acoleopteran active plant non-specific lipid acyl hydrolase transgenestably incorporated into its genome, and such progeny plants willinherit the traits afforded by the introduction of a stable transgene inMendelian fashion. All such transgenic plants having incorporated intotheir genome transgenic DNA sequences encoding any DNA constructdisclosed herein, particularly those disclosed in the examples andfigures are aspects of this invention.

[0138] Recombinant plants, cells, seeds, and other tissues could also beproduced in which only the mitochondrial or chloroplast DNA has beenaltered to incorporate the to molecules envisioned in this application.Promoters which function in chloroplasts have been known in the art(Hanley-Bowden et al., Trends in Biochemical Sciences 12:67-70, 1987).Methods and compositions for obtaining cells containing chloroplastsinto which heterologous DNA has been inserted has been described byDaniell et al., U.S. Pat. No. 5,693,507 (1997).

[0139] In another preferred embodiment, the present invention provides amethod for expressing coleopteran active plant non-specific lipid acylhydrolases at high levels in transgenic plants. The disclosed methodsmay exploit any of the DNA constructs disclosed herein, as well as anytransformation vectors known in the art. The contemplated methods enablecoleopteran active plant non-specific lipid acyl hydrolases for thecontrol of several insect pests, to be expressed in plants withoutnegatively affecting the recovery of agronomic qualities of transgenicplants. The invention described herein also enables expression ofcoleopteran active plant non-specific lipid acyl hydrolases at levels upto 10 times higher than that achieved by current methods.

[0140] The method described here thus enables plants expressingnon-specific lipid acyl hydrolase to be used as either an alternative orsupplement to plants expressing Cry1, Cry2, and Cry3-type B.thuringiensis δ-endotoxins for both control and resistance management ofkey insect pests, including Ostrina sp, Diatraea sp, Helicoverpa sp,Spodoptera sp in Zea mays; Heliothis virescens, Helicoverpa sp,Pectinophora sp. in Gossypium hirsutum; and Anticarsia sp, Pseudoplusiasp, Epinotia sp in Glycine max. It is also contemplated that the methodsdescribed may be used to dramatically increase expression of plantnonspecific lipid acyl hydrolases including and related to potatopatatin or homologues thereof, or permuteins thereof, thus increasingits effectiveness against target pests and decreasing the likelihood ofevolved resistance to these proteins. In one embodiment of the presentinvention, the coleopteran active plant non-specific lipid acylhydrolase is expressed.

[0141] The method of expressing a coleopteran active plant non-specificlipid acyl hydrolase in a plant disclosed herein includes the steps of:(1) obtaining nucleic acid sequence comprising a promoter operablylinked to a first polynucleotide sequence encoding a signal peptide fortargeting a protein to a type II secretory apparatus, and a secondpolynucleotide sequence, encoding a coleopteran active plantnon-specific lipid acyl hydrolase, to yield a fusion protein comprisedof an amino-terminal type II signal peptide and a coleopteran activeplant non-specific lipid acyl hydrolase; and (2) transforming the plantwith the DNA construct of step 1 so that the plant expresses the proteinfusion. In a preferred embodiment, the nucleic acid segment employed instep (1) of this method is structured so that the 5′ end of the secondpolynucleotide sequence is operably linked in the same translationalreading frame to the 3′ end of the first polynucleotide sequence.

[0142] The plant or plant cell transformed by the method disclosedherein may be either a monocotyledonous plant or a dicotyledonous plant.Where the plant is a monocotyledonous plant, it may be any one of avariety of species. Preferred monocotyledonous species encompassed bythe present invention may include maize, rice, wheat, barley, oats, rye,millet, sorghum, sugarcane, asparagus, turfgrass, or any of a number ofother grains or cereal plants. In preferred embodiments, the monocot isa maize plant.

[0143] The present invention also contemplates a process by which avariety of dicotyledonous plants or plant cells are transformed. Suchdicotyledonous plants may include plants such as cotton, soybean,tomato, potato, citrus, tobacco, sugar beet, alfalfa, fava bean, pea,bean, apple, cherry, pear, strawberry, raspberry, or any other legume,tuber, or fruit plant. In preferred embodiments, the dicot is a soybeanplant, a tobacco plant or cell, or a cotton plant or cell.

[0144] As noted with regard to other embodiments disclosed in thepresent invention, many of the plants intended to be transformedaccording to the disclosed invention are commercial crop plants. Thecommercial form of these plants may be the original plants, or theiroffspring which have inherited desired transgenes. Accordingly, theinventors further contemplate that the method disclosed herein includesa method of producing a transgenic progeny plant or progeny plant cell.The method of producing such progeny includes: The method of expressinga coleopteran active plant non-specific lipid acyl hydrolase in a plantdisclosed herein includes the steps of: (1) obtaining nucleic acidsequence comprising a promoter operably linked to a first polynucleotidesequence encoding a signal peptide for targeting a protein to a type IIsecretory apparatus, and a second polynucleotide sequence, encoding acoleopteran active plant non-specific lipid acyl hydrolase, to yield afusion protein comprised of an amino-terminal plastid transit peptideand a coleopteran active plant non-specific lipid acyl hydrolase; (2)obtaining a second plant; and (3) crossing the first and second plantsto obtain a crossed transgenic progeny plant or plant cell which hasinherited the nucleic acid segments from the first plant. The presentinvention specifically encompasses the progeny, progeny plant or seedfrom any of the monocotyledonous or dicotyledonous plants.

[0145] In another preferred embodiment, the method of expressing thecoleopteran active plant non-specific lipid acyl hydrolases disclosedherein includes co-expression of the disclosed DNA construct in any ofits various embodiments, along with a B. thuringiensis δ-endotoxin or aXenorhabdus sp. or Photorhabdus sp. insect inhibitory protein. Themethod of expressing these bacterial insect inhibitory proteins andhydrolases together is expected to achieve increased insect inhibitoryproperties in the transformed plant through increased expression anddecreased development of insect resistance—all of which are desiredresults not present in existing technologies. This co-expression may bein the original transformant, or in any number of generations of progenyof the original transformant which have inherited the genes toco-express the proteins encoded for by any of the DNA constructsdisclosed herein.

[0146] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLES Example 1

[0147] This example illustrates the preferred materials and methods usedin this disclosure and should not be understood to be limiting. Theexample also illustrates the DNA and amino acid sequence of Pat17 andthe variant peptides which were produced using alanine scanningmutagenesis.

[0148] Patatin is a member of a family of proteins found in potato andother solanaceous plants (Ganal, M., et al., Genetic and physicalmapping of the patatin genes in potato and tomato. Mol Gen Genetics,1991. 225: 501-509; Vancanneyt, G., to et al., Expression of apatatin-like protein in the anthers of potato and sweet pepper flowers.Plant Cell, 1989. 1: 533-540). In potatoes, patatin is predominantlyfound in tubers, and at much lower levels in other plant organs (Hofgen,R. and L. Willmitzer, Biochemical and genetic analysis of differentpatatin isoforms expressed in various organs of potato (Solanumtuberosum). Plant Sci., 1990. 66:221-230). Genes that encode patatinshave been previously isolated and characterized (Mignery, G. A., et al.,Isolation and sequence analysis of cDNAs for the major potato tuberprotein, patatin. Nucleic Acids Research, 1984. 12:7987-8000; Mignery,G. A., C. S. Pikaard, and W. D. Park, Molecular characterization of thepatatin multigene family of potato. Gene, 1988. 62:27-44; Stiekema, W.J., et al., Molecular cloning and analysis of four potato tuber mRNAs.Plant Mol Biol, 1988. 11:255-269). These proteins have been shown tohave acyl-hydrolase activity that catalyzes the non-specific hydrolysisof phospholipids, glycolipids, sulfolipids, and mono- anddiacylglycerols (Hirayama, O., et al., Purification and properties of alipid cyl-hydrolase from potato tubers. Biochim Biophys Acta, 1975.384:127-137; Wardale, D. A., Lipid-degrading enzymes from potato tubers.Phytochemistry, 1980. 19:173-177). In addition, patatin has been shownto have insect inhibitory activity against corn rootworm, aneconomically important insect pest in corn (Strickland, J. A., G. L.Orr, and T. A. Walsh, Inhibition of Diabrotica larval growth by patatin,the lipid acyl hydrolase from potato tubers. Plant Physiol, 1995.109:667-674). The current treatment used to control insect pests,including lepidopteran and coleopteran species, is δ-endotoxins ofBacillus thuringiensis (Bt) (English, L., et al., Modulation ofdelta-endotoxin ion channels. Molecular action of insecticides on ionchannels, ed. J. M. Clark. Vol. 591. 1995: Amer. Chem. Soc. Symposium.302-307; Schnepf, E., et al., Bacillus thuringiensis and its pesticidalcrystal proteins. Microbiology and molecular biology reviews, 1998.62:775-806; Crickmore, N., et al., Revision of the nomeclature for theBacillus thuringiensis pesticidal crystal proteins. Microbiology andMolecular Biology Reviews, 1998. 62:807-813). The mechanism of action ofBt proteins involves insertion of the toxin into the membrane of theinsect midgut to create ion channels or pores (English et al., ibid;Schnepf et al., ibid). Because of the widespread use of Bt toxins, thereis concern that development of resistance can shorten their usefulproduct life. Laboratory selection has produced many resistant insectsto Bt protein, but to date there is only one insect, diamondback moth(Plutella xylostella), that has evolved substantial resistance in thefield (Tabashnik, B. E., et al., Cross-resistance of the diamondbackmoth indicates altered interactions with domain II of Bacillusthuringiensis toxins. Applied and Environmental Microbiology, 1996.62:2839-2844). Patatins afford a different gene product for control ofinsect pests with a different mode of action which can be combined withBt δ-endotoxins for resistance management.

[0149] A potato cDNA gene encoding an isozyme of patatin, designatedherein as Pat17, was isolated from total DNA of Solanum cardiophyllumtubers as described herein and sequenced. The nucleotide (SEQ ID NO:37)and amino acid (SEQ ID NO:1) sequence of Pat 17 is shown in FIG. 1.Comparison of this sequence with other lipases indicated that Pat17 hadthe conserved amino acid motif (Gly-Xxx-Ser-Xxx-Gly) describingesterases (Mignery et al (1), ibid; Mignery et al (2), ibid; Steikma etal, ibid; Rosahl, S., et al., Isolation and characterization of a genefrom Solanum tuberosum encoding patatin, the major storage protein ofpotato tubers. Mol Gen Genet, 1986. 203:214-220). Chemical modificationstudies of patatin using diisopropyl fluorophosphate (DFP) eliminatesboth the enzymatic and insect inhibitory activities (Strickland et al.,ibid). Based on chemical modification experiments and the priordisclosure of Walsh et al., (U.S. Pat. No. 5,743,477), Ser77 asimplicated as being within the hydrolase motif and was solelyresponsible for the hydrolase activity and insect inhibitorybioactivity. However, other acyl hydrolase proteins had been observed tohave a catalytic triad composed of Ser, Asp/Glu and His as a part oftheir active sites and so it was postulated that patatin may alsocontain other residues responsible for activity (Strickland et al.,ibid; Senda, K., et al., A cytosolic phospholipase A2 from potatotissues appears to be patatin. Plant Cell Physiol, 1996. 37:347-353;Schrag, J. D., et al., Ser-His-Glu triad forms the catalytic site of thelipasefrom Geotrichum candidum. Nature, 1991. 351:761-764).

[0150] Therefore, alanine-scanning mutagenesis was used to identify anylikely catalytic residues (Cunningham, B. and J. Wells, High-ResolutionEpitope Mapping of hGH-Receptor Interactions by Alanine-ScanningMutagenesis. Science, 1989. 244:1081-1085; Bennett, W. F., et al., Highresolution analysis of functional determinants on human tissue-typeplasminogen activator. J Biol Chem, 1991. 266:5191-5201). All codons inthe Pat17 coding sequence encoding charged residues were altered toencode alanine in groups of 1-3 residues (Table 1). The “charged toalanine” scan variants would also help to identify residues, in additionto potential catalytic residues, which are important for activity and/orstability. A set of 75 variants were constructed using site-directedmutagenesis as shown in Table 1. All the variants were expressed inPichia pastoris and assayed for enzyme activity. The variants with verylow enzyme activity were subsequently purified and assayed forbioactivity. Based on the consensus esterase motif,Gly-Xxx₁-Ser-Xxx₂-Gly, we also changed the codon for Ser77 to Ala77 toverify that this residue is indeed responsible for catalytic andbioactivity. The inventors herein show that Pat 17 contains serine andaspartate residues that are critical for both enzymatic and insectinhibitory activities. In addition, the inventors herein have identifieda histidine residue at position 109 as important in maintaining enzymestability. The results herein suggest that Pat17 is similar to arecently identified phospholipase A₂ also employing a serine/aspartatedyad in catalysis (Dessen, A., et al., Crystal structure of humancytosolic phospholipase A ₂ reveals a novel topology and catalyticmechanism. Cell, 1999. 97:349-360). Surprisingly, however, the Dessen etal. amino acid sequence fails to align at all with any of the plantderived sequences disclosed herein, indicating only that the twoproteins contain active sites based on a similar biocatalytic theme butwhich exhibit substantially unrelated sequences and activitiesotherwise. TABLE 1 Charged to Alanine Scan Variants Column 1 Column 2Wild type D223A E27A R234A D35A K238A R40A D239A E49A R246A E52AK251A/K252A E57A/D59A E265A/D267A D63A K268A R65A K273A D68A E274A D71AH282A S77A K289A E91A D292A R94A D300A K100A D311A E101A K313A E108AR318A H109A E321A K124A E330A D126A D332A K128A D333A E136A E336A K137AE340A E140A E347A R142A K351A/K352A H144A E356A/D357A E149A E360A D156AE363A K158A E364A K161A K367A K167A R368A E175A K371A D177A D375A K179AR376A D182A K377A H197A K378A D207A/E208A/E210A R380A D215A K383A

[0151] Genes for patatin have been cloned by several investigators, asindicated above. The sequence disclosed was used to design primers toclone the Pat17 gene from S. cardiophyllum. Total RNA was prepared fromSolanum cardiophyllum tubers using TRI REAGENT according to themanufacturers protocol (Molecular Research Center, Inc.). The RNA wasused to generate cDNA using reverse transcription. A full-length cDNA ofPat 17 was amplified using thermal ampification methods and theamplification primers SEQ ID NO:185′-GTTAGATCTCACCATGGCAACTACTAAATCTTT-3′ (NcoI site indicated byunderlined bases) and SEQ ID NO:195′-CCAGAATTCTCATTAATAAGAAGCTTTGTTTGC-3′ (EcoRI site indicated byunderlined bases).

[0152] Standard thermal amplification reaction conditions as describedin the GENE AMP kit (Perkin-Elmer Cetus) were used, however an annealingtemperature of 40° C. was used in the alternative. Resulting DNA wascloned into pBluescript plasmid (Stratagene, CA) and the insert wasconfirmed by DNA sequence analysis.

[0153] Pat17 variants were generated using an oligonucleotide-directedmutagenesis protocol from Bio-Rad Laboratories (Richmond, Calif.) whichis based on the method of Kunkel (Kunkel, D. A., Rapid and efficientsite-specific mutagenesis without phenotypic selection. Proc Natl AcadSci USA, 1985. 82:477-92). The Pat17 gene was cloned into the plasmidpBluescript SK+ (Strategene, CA) under conditions which facilitated thegeneration of single-stranded DNA. The mutagenesis procedure wasfollowed as outlined in the protocol. Mutagenic oligonucleotides werepurchased from Midland Reagent Company (Midland, Tex.). Mutant cloneswere identified by sequencing the region covered by the mutagenicoligonucleotides.

[0154] The wild-type and Pat 17 variants were digested with XhoI/EcoRIand ligated to the respective sites in the P. pastoris expression vectorpPIC9 (Invitrogen, CA) used for extracellular expression. Thetransformation of the P. pastoris strain KM71 (Invitrogen, CA),screening for recombinants, and expression experiments were performed asoutlined according to the manufacturer's instructions.

[0155] Culture supernatants of P. pastoris transformants producingrecombinant protein were dialyzed against 25 mM Tris/HCl pH 7.5 (bufferA) and loaded onto Mono Q HR 10/10 anion-exchange column (AmershamPharmacia, NJ) equilibrated with buffer A. The protein was eluted with25 mM Tris/HCl pH 7.5, 1 M KCl (buffer B) using a linear gradient of0-100% buffer B run over 30 min at a flow rate of 4 mL/min using an HPLCsystem (Shimadzu). Fractions containing protein were assayed foresterase activity, dialyzed against 25 mM Tris/HCl pH 7.5, 1 M Ammoniumsulfate, 1 mM β-mercaptoethanol (buffer C). The protein was purified tohomogeneity by loading onto a phenyl-Sepharose 16/10 column (AmershamPharmacia, NJ) equilibrated with buffer C. The protein was eluted withbuffer A using a linear gradient of 0-100% at a flow rate of 3 mL/minusing an HPLC system (Shimadzu). Esterase active fractions were pooledand dialyzed against 25 mM Tris pH 7.5.

[0156] Enzyme activity was measured as described previously usingp-nitrophenyl caprate (Sigma, MO) as a substrate (Hofgen et al., ibid).The substrate was initially dissolved in dimethylsulfoxide (5 mM stocksolution) and diluted in 4% Triton X-100, 1% SDS to a finalconcentration of 1 mM. For the assay, 25 μL of the 1 mM substratesolution was added to 80 μL of 50 mM Tris pH 8.5 prior to the additionof 20 μL of protein solution. The enzyme activity was monitored at 405nm in 6 sec interval for a period of 10 min. Esterase activity wasexpressed as ΔA min⁻¹ ug⁻¹ protein. Steady-state kinetic assays atdifferent pH's were performed using Sodium acetate (pH 4-5.0), MES (pH5-7.0), TRIZMA (pH 7-9.0), CHES (pH 9.5) with a 150 μL total volume.Assays were initiated with 10 μL of enzyme containing 0.1 mg/mL proteinin 25 mM Tris pH 7.5. The reactions were quenched after 5 min with 850μL of 200 mM Borate buffer (pH 9.8) and the absorbance was measured at405 nm. The reaction rate was calculated using an extinction coefficientof 18.4 for p-nitrophenol. The K_(m) values for the substrate wasdetermined by varying the substrate concentration (5-10 time the K_(m)value). The steady-state kinetic data were analyzed using KINETASYST(IntelliKinetics, NJ).

[0157] Insect bioassays for activity against larvae of Diabroticaundecimpunctata howardi (southern corn rootworm) were carried out byoverlaying the test sample on an agar diet similar to that describedpreviously (Marrone, P., et al., Improvements in laboratory rearing ofthe southern corn rootworm, Diabrotica undecimpuncta howardi barber(coleoptera: chrysomelidae), on an artificial diet and corn. J. Econ.Entom., 1985. 78:290-293). Proteins to be tested were diluted in 25 mMTris/HCl pH 7.5 and overlayed on the diet surface. Neonate larvae wereallowed to feed on the diet and mortality and growth stunting wereevaluated after 6 days.

[0158] N-terminally-His-tagged Seleno-Methionine (Se-Met) Pat17 wasexpressed by metabolic labeling with Se-Met in a Se-Met-tolerant Metauxotroph of E. coli and was purified using Ni-chelate followed by anionexchange chromatography. Electrospray mass spectrometry revealed thatthe enzyme sample (41833 Da) contained Se-Met residues at all 13methionine positions. The enzyme was crystallized using the technique ofvapor diffusion by hanging drops. The protein sample was 10 mg/ml in 10mM Tris-pH 7.4 and the precipitant solution was 16% PEG3350, 0.24 Mammonium acetate. A droplet comprised of 2 ml of protein solution and 2ml of precipitant solution were placed on a siliconized coverslip andsuspended over a grease-sealed well of a Linbro plate containing 500 mlof precipitant solution. Crystals appeared within five days. Preliminaryin-house diffraction analyses on cryo-cooled crystals were conductedusing an MSC R-AXIS IV imaging plate detector mounted on an MSC RU300H3RX-ray generator, operating at a power of 50 kV and 100 mA, with beamcollimation provided by MSC/Yale mirrors, and cryo-cooling achievedusing an MSC X-Stream unit operating at approximately −140 degrees C.Crystals taken from the drops were dipped in a cryo-solution which was16.5% PEG3350, 0.23 M ammonium acetate, 25% ethylene glycol prior toflash-cooling in the cold stream of the R-AXIS IV unit. Diffractionstudies revealed that the crystals were space group C222₁, with a=97.2Å, b=171.4 Å, c=129.8 Å, and that they diffracted to better than 2.5 Åresolution. Protein/solvent content calculations based on the latticeand diffraction quality of the crystals suggested three Pat17 moleculesin the asymmetric unit. The structure was solved using Se-MetMulti-wavelength Anomalous Dispersion (MAD) phasing methods. Fourwavelengths of MAD data (11=0.9791 Å, 12=0.9792 Å, 13=1.019 Å, 14=0.942Å) were collected at the IMCA beamline of the APS synchrotron. AMarresearch CCD detector was used to collect the diffraction data andthe crystal was cryo-cooled using the aforementioned cryo-solution andan Oxford Cryo-stream unit operating at approximately −140 degrees C.360 degrees of data at each wavelength were collected using 2.5 secondexposures, an oscillation angle of 0.5 degrees, and acrystal-to-detector distance of 130 mm. The data were reduced using theHKL2000 package. The SOLVE program was employed to locate 33 of 39 Sesites in the asymmetric unit using 20-2.2 Å data. Phases from SOLVE wereimproved using the CCP4 package utility DM. A single Pat17 molecule wasbuilt into a 2.2 Å resolution experimental map using an SGI Octaneworkstation with stereo-graphics capability, the O program and theInsightII Biopolymer module. The Pat 17 coordinates, 8-3.5 Å data, andthe AMoRe molecular replacement package were used to locate all threemolecules in the asymmetric unit (R−f=0.384).

Example 2

[0159] This example illustrates the lipid acyl hydrolase esteraseactivity of the charged to alanine scan variants described in Example 1.

[0160] Table 1 shows the list of charged to alanine scan variants. Allthe variants were expressed in P. pastoris and assayed for esteraseactivity as shown in FIG. 2. The level of protein expression was assayedusing an ELISA and a monoclonal antibody specific for the Pat17 nativeamino acid sequence. Some of the variants could not be expressedincluding E52A, D68A, D71A and H109A, suggesting that these residues arecritical for enzyme stability. Variants E91A, R94A and E136A showed goodenzyme activity but could not be detected by the monoclonal antibodyused in the ELISA suggesting that these are the potential recognitionepitopes for the monoclonal antibody. All variants were assessed onWestern blots probed with a polyclonal antibody to validate the ELISAexpression values. The variant comprising D215A showed significant lossin esterase activity suggesting that this residue is critical foresterase activity (FIG. 2 and Table 2). TABLE 2 Esterase Activity fVariants at Position 77, 109 and 215. Esterase Activity Variants (ΔOD ·min⁻¹ · μg⁻¹) Wild type 116.0 S77A 0.02 S77D 0.01 S77T 0.1 S77N 0.01S77C 0.1 S77R^(a) N/A H109A^(a) N/A H109N 234.5 D215A 0.02

[0161] As Ser77 lies in a hydrolase motif identified in U.S. Pat. No.5,743,477, a S77A variant was constructed to elucidate its role incatalysis. As shown in FIG. 2, S77A was inactive towards the esterasesubstrate, suggesting that this residue is necessary for catalysis.Activity greater than that of the wild type Pat17 was observed for thevariants at positions 65 and 352 (5-fold increase). Based on the X-raycrystal structure, the side chains of these basic residues (R65A,K351A/K352A) appear to lie 10 on surface loops and to be facing in thesame direction. Esterase activity of all the other variants varied from0.5-fold to 4.2-fold respectively of the wild type protein. Severalvariants were also made at position 77 including S77A, S77D, S77T, S77N,S77C and S77R in order to elucidate the primary sequence requirementsfor enzymatic activity. The results of the esterase activity assay forthe variants at position 77 are shown in Table 2. All the Ser77 variantswere found to be inactive towards esterase substrates compared to thewild type enzyme suggesting that Ser77 is one of the catalytic residueinvolved in covalent catalysis. Histidine is usually a very conservedresidue in the normal lipase catalytic triad, and thus we changed His109to asparagine (an isosteric residue to His) and evaluated its esteraseactivity (shown in Table 2). It was surprising to note that H109Nmaintained full catalytic activity. Other changes at this positionincluding H109C, H109D, H109R could not be expressed suggesting that thenitrogen atom in His109 is critical for maintaining the activity of theenzyme. This result rules out the possibility that His109 plays a directrole in catalysis. This data is further supported by the X-ray crystalstructure which shows that His109 stabilizes the interaction between twohelices and probably helps in maintaining the overall conformation ofthe protein.

Example 3

[0162] This example illustrates the pH rate profile of the native Pat17enzyme.

[0163] The plot of the data for k_(cat)/K_(m) for p-nitrophenyl capratesubstrate is shown in FIG. 3. The pH-independent value of the kineticparameters are: k_(cat)=2.7 s⁻¹ and k_(cat)/K_(m)=9.3 mM⁻¹ s⁻¹. Thek_(cat)/K_(m) is essentially pH independent over the pH range of 5-9.5.This result suggests that a single residue with a pKa <5 must bedeprotonated for enzyme activity, supporting the alanine scanningmutagenesis which identified Asp215 as at least one of the catalyticresidues.

Example 4

[0164] This example illustrates the coordinated requirement forfunctional enzyme activity and insect inhibition for the native andvariant forms of patatin.

[0165] It has previously been shown that the enzymatic activity ofpatatin is required for it to also display effective insect inhibitorybioactivity. Therefore, the Ser77 variants described above (S77A, S77D,S77T, S77N, S77C) and the aspartate variant D215A were tested in aninsect bioassay against southern corn rootworm (SCRW). The results areshown in FIG. 4. All of the assays were performed by overlaying protein(200 ppm final concentration) onto a corn rootworm artificial dietmedium. All insects growth was stunted when native Pat17 was used,however no insect mortality was observed. All esterase inactive variantsdisplayed no activity against SCRW suggesting that Ser77 and Asp215 arerequired for esterase activity and insect inhibitory bioactivity.

[0166] Assays were also conducted to evaluate the bioactivity of theH109N variant. As shown in FIG. 5, H109N had similar activity as thewild type enzyme in inhibiting the growth of SCRW larvae. The assay forH109N was performed in a similar manner as the other assays but thefinal concentration of overlayed protein was 100 ppm.

Example 5

[0167] This example illustrates the model for the chemical mechanism ofpatatin non-specific lipid acyl hydrolase catalysis.

[0168] Patatin has been classified as a Ser hydrolase due to thepresence of the general amino acid motif, Gly-Xxx₁-Ser-Xxx₂-Gly in theprotein sequence. Previous chemical modification studies have shown thatDFP-treated patatin had >20-fold reduction in esterase activity and nobioactivity. The instant disclosure describes the cloning of an isozymeof patatin designated herein as Pat17. On the basis of theGly-Xxx₁-Ser-Xxx₂-Gly consensus sequence, Ser77 is predicted to beinvolved in catalysis in Pat17. As the structure of patatin was notknown when this work was initiated, other catalytic residues in the α/βhydrolase fold family of enzymes were also implicated. As in the familyof α/β hydrolases, the nucleophile can either be Ser, Cys or Asp.Therefore, the inventors herein altered the Ser77 to Ala, Cys, Asp, Thr,Asn, and Arg. All the variants were assayed for esterase and insectinhibitory activity and the results indicate that this residue iscritical for both activities. Patatin has also been classified as alipid acyl hydrolase because it exhibits phospholipase activity. Thesequential order of active site residues in some lipases is Ser,Asp/Glu, His with the Ser being the only residue identifiable bysequence gazing. Since there is no consensus motif to identify orpredict the His and the carboxylate residues, the inventors hereinutilized site-directed mutagenesis to construct a synoptic set ofclustered point mutations in Pat17 by changing all the charged residuesin the protein including Glu, Asp, His, Lys, and Arg to alanine ingroups of 1-3 to identify the active site residues. This method,“clustered charged-to-alanine scan,” has previously been used toidentify critical residues in other proteins. The results describedherein have identified Asp215 as the carboxylate residue critical forcatalysis. The pH rate profile of Pat17 reveals that an acidic groupwith a pKa of <5 is important in catalysis suggesting that Asp215 withinthe Glu-Xaa₁-Xaa₂-Leu-Val-Asp-Gly consensus motif is the catalytic base(FIG. 3). The X-ray crystal structure indicates that Ser77 and Asp215are within hydrogen bonding distance and thus support the notion thatthese residues are the catalytic residues (FIG. 6a,b). The resultsherein also suggest that His 109 is critical for maintaining theactivity of the enzyme. The substitution of Ala, Cys, Asp, or Arg atposition 109 is not permitted as no protein could be detected by ELISAand/or Western blot, suggesting that this position might be crucial forstability of the enzyme. An isosteric change at this position (H109N)generates a protein which maintains full esterase and insect inhibitoryactivity. An analysis of the patatin homolog alignment in FIG. 9indicates that the Histidine or Asparagine at this position is alsowithin a conserved sequence as set forth in SEQ ID NO:42 asPhe-Tyr-Xaa₁-Glu-His/Asn-Gly-Pro, wherein the Xaa₁ can be either Phe,Ile, or Leu.

[0169] Analysis of the X-ray crystal structure indicates that His109stabilizes the interaction between two helices by acting as a nucleus ofa hydrophobic/polar cavity bounded by Phe105, Glu108, Ile113, Tyr129,Val133 and Lys137 (FIG. 7). This residue probably helps stabilize thestructure by keeping the helices in close proximity and thus helps tomaintain the overall fold of the enzyme. An asparagine at position 109(H109N variant), maintains full esterase and bioactivity. All of thedata discussed supports the roles of Ser77 and Asp215 as criticalresidues in catalysis which is also supported by the pH profile and theX-ray crystal structure. In addition, two variants at positions 65 and252 (R65A, K251A/K252A) have also been identified which exhibited a5.0-fold increase in esterase activity compared to the wild type enzyme.Examining the crystal structure reveals that these residues arepredicted to be located at the Pat17 molecular surface. Further analysiscan be done to assess their role in insect inhibition. Charged toalanine substitutions has previously been used to generate variants withincreased specificity for substrates.

[0170] A model depicting the roles of Ser77 and Asp215 in catalysis isillustrated in FIG. 8. This model illustrates that Ser77 can serve asthe nucleophile that attacks the carbonyl carbon of the scissile peptidebond with Asp215 serving as the base. This is supported by X-ray crystalstudies which indicate that Ser77 and Asp215 lie within hydrogen bondingdistance from each other and they make up the elements of the activesite (FIG. 6a,b).

[0171] The model depicted herein suggests that patatin uses a Ser-Aspdyad rather than the standard Ser-His-Asp triad found in proteases,lipases and esterases. Recently, a phospholipase A₂ has been identifiedthat has a similar Ser-Asp dyad in the active site. The results hereinsuggest that patatin is a member of a new family of lipid acylhydrolases that employ Ser-Asp dyad in catalysis. Recently, other novelserine proteases have been discovered that use hydroxyl/ε-amine orhydroxyl/α-amine catalytic dyads to perfom catalysis. The identificationof a new class of lipid acyl hydrolases that utilize Ser-Asp catalyticdyads, depicted by patatin and phospholipase A₂, suggest that othervariations in the classical catalytic triad theme in addition to theSer/Lys catalytic dyads exist, and further structure/function studies ofthese enzymes would lead to a better understanding of these proteins.

Example 6

[0172] This example illustrates the construction and analysis ofpermuteins of patatin and patatin homologues. Nucleic acid sequencesencoding permutein proteins having rearranged N-terminus/C-terminusprotein sequences can be made by following the general method describedby Mullins et al. (J. Am. Chem. Soc. 116: 5529-5533, 1994). The stepsare shown in FIG. 10, and this example involves the design and use of alinker region separating the original C-terminus and N-terminus, but theuse of a linker is not a critical or required element of permuteindesign.

[0173] Two sets of oligonucleotide primers are used in the constructionof a nucleic acid sequence encoding a permutein protein. In the firststep, oligonucleotide primers “new N-termini” and “linker start” areused in a PCR reaction to create amplified nucleic acid molecule “newN-termini fragment” that contains the nucleic acid sequence encoding thenew N-terminal portion of the permutein protein, followed by thepolypeptide linker that connects the C-terminal and N-terminal ends ofthe original protein. In the second step, oligonucleotide primers “newC-termini” and “linker end” are used in a PCR reaction to createamplified nucleic acid molecule “new C-termini fragment” that containsthe nucleic acid sequence encoding the same linker as used above,followed by the new C-termini portion of the permutein protein. The “newN-termini” and “new C-termini” oligonucleotide primers are designed toinclude appropriate restriction enzyme recognition sites which assist inthe cloning of the nucleic acid sequence encoding the permutein proteininto plasmids.

[0174] Any suitable PCR conditions and polymerase can be used. It isdesirable to use a thermostable DNA polymerase with high fidelity toreduce or eliminate the introduction of sequence errors. Typical PCRconditions are 25 cycles 94° C. denaturation for 1 minute, 45° C.annealing for one minute and 72° C. extension for 2 minutes; plus onecycle 72° C. extension for 10 minutes. A 50 μL reaction contains 30 pmolof each primer and 1 μg of template DNA; and 1×PCR buffer with MgCl₂,200 μM dGTP, 200 μM dATP, 200 μM dTTP, 200 μM dCTP, 2.5 units of Pwo DNApolymerase. PCR reactions are performed in RoboCycler Gradient 96Temperature Cycler (Stratagene, La Jolla, Calif.).

[0175] The amplified “new N-termini fragment” and “new C-terminifragment” are annealed to form a template in a third PCR reaction toamplify the full-length nucleic acid sequence encoding the permuteinprotein. The DNA fragments “new N-termini fragment” and “new C-terminifragment” are resolved on a 1% TAE gel, stained with ethidium bromide,and isolated using the QIAquick Gel Extraction Kit (Qiagen, Valencia,Calif.). These fragments are combined in equimolar quantities witholigonucleotide primers “new N-termini” and “new C-termini” in the thirdPCR reaction. The conditions for the PCR are the same as usedpreviously. PCR reaction products can be purified using the QIAquick PCRpurification kit (Qiagen, Valencia, Calif.).

[0176] Alternatively, a linker sequence can be designed containing arestriction site, allowing direct ligation of the two amplified PCRproducts.

[0177] Construction of Plasmid pMON 37402

[0178] The patatin protein contains a trypsin protease sensitive site atthe arginine amino acid at position 246, as determined byelectrophoresis of a trypsin digest reaction. In order to determine ifthe exposed protease site is an antigenic epitope, a permutein wasconstructed using positions 246-247 as a breakpoint.

[0179] The nucleic acid sequence encoding the permutein protein inplasmid pMON 37402 was created using the method illustrated in FIG. 10and described herein. Nucleic acid molecule “new N-termini fragment” wascreated and amplified from the sequence encoding patatin in plasmidpMON26820 using oligonucleotide primers 27 (SEQ ID NO:242 SEQ ID NO:43)and 48 (SEQ ID NO:243 SEQ ID NO:44). Nucleic acid molecule “newC-termini fragment” was created and amplified from the sequence encodingpatatin in plasmid pMON26820 using oligonucleotide primers 47 (SEQ IDNO:244 SEQ ID NO:45) and 36 (SEQ ID NO:245 SEQ ID NO:46). Thefull-length nucleic acid molecule encoding the permutein protein wascreated and amplified from annealed fragments “new N-termini fragment”and “new C-termini fragment” using oligonucleotide primers 27 (SEQ IDNO:242 SEQ ID NO:43) and 36 (SEQ ID NO:245 SEQ ID NO:46).

[0180] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

[0181] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm to the presence of the correct insert.The resulting plasmid was designated pMON 37402 (containing SEQ IDNO:20, encoding protein sequence SEQ ID NO:21).

[0182] Construction of Plasmid pMON 37405

[0183] Amino acids 201-202, near tyrosine 193, were chosen as abreakpoint for the construction of a permutein protein.

[0184] The nucleic acid sequence encoding the permutein protein inplasmid pMON 37405 was created using the method illustrated in FIG. 10and described herein.

[0185] Nucleic acid molecule “New N-termini fragment” was created andamplified from the sequence encoding patatin in plasmid pMON26820 usingoligonucleotide primers 48 (SEQ ID NO:44) and 58 (SEQ ID NO:47). Nucleicacid molecule “New C-termini fragment” was created and amplified fromthe sequence encoding patatin in plasmid pMON26820 using oligonucleotideprimers 47 (SEQ ID NO:45) and 59 (SEQ ID NO:47). The full-length nucleicacid molecule encoding the permutein protein was created and amplifiedfrom annealed fragments “New N-termini fragment” and “New C-terminifragment” using oligonucleotide primers 58 (SEQ ID NO:48) and 59 (SEQ IDNO:47).

[0186] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

[0187] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON 37405 (containing SEQ IDNO:22, encoding protein sequence SEQ ID NO:23).

[0188] Construction of Plasmid pMON 37406

[0189] Amino acids 183-184, adjacent to tyrosine 185, were chosen as abreakpoint for the construction of a permutein protein.

[0190] The nucleic acid sequence encoding the permutein protein inplasmid pMON 37406 was created using the method illustrated in FIG. 10and described herein. Nucleic acid molecule “New N-termini fragment” wascreated and amplified from the sequence encoding patatin in plasmidpMON26820 using oligonucleotide primers 48 (SEQ ID NO:44) and 60 (SEQ IDNO:49). Nucleic acid molecule “New C-termini fragment” was created andamplified from the sequence encoding patatin in plasmid pMON26820 usingoligonucleotide primers 47 (SEQ ID NO:45) and 61 (SEQ ID NO:50). Thefull-length nucleic acid molecule encoding the permutein protein wascreated and amplified from annealed fragments “New N-termini fragment”and “New C-termini fragment” using oligonucleotide primers 60 (SEQ IDNO:49) and 61 (SEQ ID NO:50).

[0191] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

[0192] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON37406 (containing SEQ ID NO:24,encoding protein sequence SEQ ID NO:25).

[0193] Construction of Plasmid pMON 37407

[0194] Amino acids 268-269, adjacent to tyrosine 270, were chosen as abreakpoint for the construction of a permutein protein.

[0195] The nucleic acid sequence encoding the permutein protein inplasmid pMON 37407 was created using the method illustrated in FIG. 10and described herein. Nucleic acid molecule “New N-termini fragment” wascreated and amplified from the sequence encoding patatin in plasmidpMON26820 using oligonucleotide primers 48 (SEQ ID NO:44) and 62 (SEQ IDNO:51). Nucleic acid molecule “New C-termini fragment” was created andamplified from the sequence encoding patatin in plasmid pMON26820 usingoligonucleotide primers 47 (SEQ ID NO:45) and 63 (SEQ ID NO:52). Thefull-length nucleic acid molecule encoding the permutein protein wascreated and amplified from annealed fragments “New N-termini fragment”and “New C-termini fragment” using oligonucleotide primers 62 (SEQ IDNO:51) and 63 (SEQ ID NO:52).

[0196] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

[0197] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON37407 (containing SEQ ID NO:26,encoding protein sequence SEQ ID NO:27).

[0198] Construction of Plasmid pMON 37408

[0199] Amino acids 321-322, near tyrosine 216, were chosen as abreakpoint for the construction of a permutein protein.

[0200] The nucleic acid sequence encoding the permutein protein inplasmid pMON 37408 was created using the method illustrated in FIG. 10and described herein. Nucleic acid molecule “New N-termini fragment” wascreated and amplified from the sequence encoding patatin in plasmidpMON26820 using oligonucleotide primers 48 (SEQ ID NO:44) and 64 (SEQ IDNO:53). Nucleic acid molecule “New C-termini fragment” was created andamplified from the sequence encoding patatin in plasmid pMON26820 usingoligonucleotide primers 47 (SEQ ID NO:45) and 65 (SEQ ID NO:54). Thefull-length nucleic acid molecule encoding the permutein protein wascreated and amplified from annealed fragments “New N-termini fragment”and “New C-termini fragment” using oligonucleotide primers 64 (SEQ IDNO:53) and 65 (SEQ ID NO:54).

[0201] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

[0202] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON37408 (containing SEQ ID NO:28,encoding protein sequence SEQ ID NO:29).

[0203] Production of Permutein Proteins in Pichia pastoris

[0204] Plasmids pMON37402, pMON37405, pMON37406, pMON37407, andpMON37408 were individually used to electroporate KM71 cells from Pichiapastoris according to the procedure supplied by the manufacturer(Invitrogen, Carlsbad, Calif.). The resulting transformed cells wereused to produce protein in Pichia pastoris following the proceduresupplied by the manufacturer (Invitrogen, Carlsbad, Calif.).

[0205] The concentration of patatin in the culture was determined usinga patatin ELISA assay and the enzyme activity was measured using themethod of Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990). Thevariants containing multiple mutations were further purified using MonoQ and hydrophobic interaction chromatography (HIC). Each culture waspurified by first sizing on YM10 membranes (Amicon, MA) to a [>10 kDa]fraction, followed by chromatography on the Mono Q HR 10/10 column(Pharmacia, NJ). For chromatography on the Mono Q column, the sampleswere loaded on the column in 25 mM Tris pH 7.5 and eluted with agradient of 1.0 M KCl in 25 mM Tris pH 7.5. Fractions containing patatinprotein were determined using SDS-PAGE. For chromatography on the HICcolumn, the appropriate fractions were pooled and dialyzed into 1 Mammonium sulfate in 25 mM Tris pH 7.5. The dialyzed sample was thenloaded on 16/10 phenyl Sepharose column (Pharmacia, NJ) and eluted witha gradient of 25 mM Tris pH7.5.

[0206] The protein concentration was determined using the Bradfordmethod, using BSA as a standard. SDS-PAGE analysis showed that theseproteins were essentially pure. The esterase activity of the variantsare shown in Table 3. TABLE 3 Activity of permuteins enzyme BreakpointActivity (ΔOD min⁻¹ μg⁻¹) Native SEQ ID NO: 1  83.21 pMON37402 SEQ IDNO: 21 246/247 66.7 pMON37405 SEQ ID NO: 23 201/202 No expressionpMON37406 SEQ ID NO: 25 183/184 No expression pMON37407 SEQ ID NO: 27268/269 12.1 pMON37408 SEQ ID NO: 29 321/322 No expression

[0207] The activity was determined using p-nitrophenyl caprate substrateas described by Hofgen and Willmitzer (Plant Science, 66: 221-230,1990).

[0208] Insect Bioefficacy Assays

[0209] Assays for activity against larvae of SCRW are carried out byoverlaying the test sample on an agar diet similar to that described byMarrone (J. Econ. Entom. 78: 290-293, 1985). Test samples were preparedin 25 mM Tris, pH 7.5 buffer. Neonate larvae are allowed to feed on thetreated diet at 26° C., and mortality and growth stunting were evaluatedafter 5 or 6 days. The results of this assay are shown in Table 4. TABLE4 Insect bioefficacy assay Mean % Weight Protein (200 ppm) SurvivalWeight Reduction Tris buffer (control) 1.26 ± 0.3  — Wild Type 0.21 ±0.02 83 pMON37402 0.21 ± 0.03 83 pMON37407 0.32 ± 0.04 75

[0210] These data demonstrate that the growth of the SCRW larvae issimilarly reduced upon ingestion of the proteins encoded by pMON37402and pMON37407 as compared to the wild type patatin protein.

[0211] Permutein Sequences Improved for Monocot Expression

[0212] Modification of coding sequences has been demonstrated above toimprove expression of insecticidal proteins. A modified coding sequencewas thus designed to improve expression in plants, especially corn (SEQID NO:31).

[0213] Construction of pMON40701 for Monocot Expression

[0214] Plasmid pMON19767 was digested with restriction endonucleasesNcoI and EcoRI and the 1100 bp gene fragment was purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). PlasmidpMON33719 was digested with restriction endonucleases NcoI and EcoRI,and gel purified, resulting in an approximately 3900 base pair vectorfragment. The two purified restriction fragments were combined andligated using T4 DNA ligase.

[0215] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON40700. Plasmid pMON40700 wasdigested with restriction endonuclease NotI and the resulting 2200 bpDNA fragment was purified using the QIAquick PCR purification kit(Qiagen, Valencia, Calif.). Plasmid pMON30460 was digested withrestriction endonuclease NotI, and gel purified, resulting in anapproximately 4200 base pair vector fragment. The two purifiedrestriction fragments were combined and ligated using T4 DNA ligase.

[0216] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on kanamycin-containing plates. The resultingplasmid was designated pMON40701 (containing SEQ ID NO:30, encodingprotein sequence SEQ ID NO:31).

[0217] Construction of pMON40703 for Monocot Expression

[0218] The nucleic acid sequence encoding the permutein protein inplasmid pMON40703 was created using the method illustrated in FIG. 10and described herein. Nucleic acid molecule “New N-termini fragment” wascreated and amplified from the sequence encoding patatin in plasmidpMON19767 using oligonucleotide primers Syn1 (SEQ ID NO:55) and Syn2(SEQ ID NO:56). Nucleic acid molecule “New C-termini fragment” wascreated and amplified from the sequence encoding patatin in plasmidpMON19767 using oligonucleotide primers Syn3 (SEQ ID NO:57) and Syn4(SEQ ID NO:58). The full-length nucleic acid molecule encoding thepermutein protein was created and amplified from annealed fragments “NewN-termini fragment” and “New C-termini fragment” using oligonucleotideprimers Syn1 (SEQ ID NO:55) and Syn4 (SEQ ID NO:58).

[0219] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases NcoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). PlasmidpMON33719 was digested with restriction endonucleases NcoI and EcoRI,and gel purified, resulting in an approximately 3900 base pair vectorfragment. The purified restriction fragments were combined and ligatedusing T4 DNA ligase.

[0220] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON40702. Plasmid pMON40702 wasdigested with NotI, and the resulting 2200 bp DNA fragment was purifiedusing the QIAquick PCR purification kit (Qiagen, Valencia, Calif.).Plasmid pMON30460 was digested with restriction endonuclease NotI, andgel purified, resulting in an approximately 4200 base pair vectorfragment. The purified restriction fragments were combined and ligatedusing T4 DNA ligase.

[0221] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on kanamycin-containing plates. The resultingplasmid was designated pMON40703 (containing SEQ ID NO:32, encodingprotein sequence SEQ ID NO:33). Plasmid pMON40703 encodes a permuteinprotein with a “breakpoint” at positions 246/247 of the wild typepatatin protein sequence (SEQ ID NO:38). The first 23 amino acids of SEQID NO:39 are a signal peptide sequence which is cleaved in the matureprotein.

[0222] Construction of pMON40705 for Monocot Expression

[0223] The nucleic acid sequence encoding the permutein protein inplasmid pMON40705 was created using the method illustrated in FIG. 10and described herein. Nucleic acid molecule “New N-termini fragment” wascreated and amplified from the sequence encoding patatin in plasmidpMON19767 using oligonucleotide primers Syn10 (SEQ ID NO:59) and Syn2(SEQ ID NO:56). Nucleic acid molecule “New C-termini fragment” wascreated and amplified from the sequence encoding patatin in plasmidpMON19767 using oligonucleotide primers Syn3 (SEQ ID NO:57) and Syn11(SEQ ID NO:60). The full-length nucleic acid molecule encoding thepermutein protein was created and amplified from annealed fragments “NewN-termini fragment” and “New C-termini fragment” using oligonucleotideprimers Syn10 (SEQ ID NO:59) and Syn11 (SEQ ID NO:60).

[0224] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases NcoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). PlasmidpMON33719 was digested with restriction endonucleases NcoI and EcoRI,and gel purified, resulting in an approximately 3900 base pair vectorfragment. The purified restriction fragments were combined and ligatedusing T4 DNA ligase.

[0225] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON40704. Plasmid pMON40704 wasdigested with restriction endonuclease NotI, and the resulting 2200 bpDNA fragment was purified using the QIAquick PCR purification kit(Qiagen, Valencia, Calif.). Plasmid pMON30460 was digested withrestriction endonuclease NotI, and gel purified, resulting in anapproximately 4200 base pair vector fragment. The purified restrictionfragments were combined and ligated using T4 DNA ligase.

[0226] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on plates containing kanamycin. The resultingplasmid was designated pMON40705 (containing SEQ ID NO:34, encodingprotein sequence SEQ ID NO:35). Plasmid pMON40705 encodes a permuteinprotein with a “breakpoint” at positions 268/269 of the wild typepatatin protein sequence (SEQ ID NO:39). The first 23 amino acids of SEQID NO:2 are a signal peptide sequence which is cleaved in the matureprotein.

[0227] Transient Expression of Protein in Corn Leaf Protoplasts

[0228] Plasmids pMON40701, pMON40703, and pMON40705 (all containing thenative signal sequence for vacuolar targeting) were separatelyelectroporated into corn leaf protoplasts as described by Sheen (PlantCell 3: 225-245, 1991). Protein was extracted with glass beads and thesupernatant was assayed for protein expression using ELISA for patatinand NPTII. Expression of protein by the transformed corn protoplasts wasconfirmed by Western blot analysis. Expression results are shown inTable 5. TABLE 5 ELISA data Normalized Patatin NPTII Expression ELISAELISA (Patatin ELISA/ enzyme (μg/mL) (μg/mL) NPTII ELISA) pMON40701 1.10.6 1.8 SEQ ID NO: 31 pMON40703 2.1 0.3 7.0 SEQ ID NO: 33 pMON40705 1.30.6 2.2 SEQ ID NO: 35

Example 7

[0229] This example illustrates the positions of critical amino acidresidues in patatin and homologs. TABLE 6 Positions of Critical AminoAcid Residues in Patatin and Homologs Catalytic Residue Other Enzyme SerAsp His/Arg Pat17 77 215 109 PatFm 55 194 87 PatIm 55 193 87 PatL+ 77215 109 PatA+ 77 215 109 PatB+ 77 215 109 Pentin1 82 222 116 5C9 72 223104 Corn 3 72 223 104 Corn 2 72 223 104 Corn 4 72 223 104 Corn 1 108 260140 Corn 5 72 223 104

[0230] In view of the above, it will be seen that the several advantagesof the invention are achieved and other advantageous results attained.

[0231] As various changes could be made in the above methods andcompositions without departing from the scope of the invention, it isintended that all matter contained in the above description, and shownin the accompanying drawings, shall be to interpreted as illustrativeand not in a limiting sense.

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1 60 1 386 PRT Solanum cardiophyllum Protein (1)..(386) patatin homologpat17 amino acid sequence 1 Met Ala Thr Thr Lys Ser Phe Leu Ile Leu IlePhe Met Ile Leu Ala 1 5 10 15 Thr Thr Ser Ser Thr Phe Ala Gln Leu GlyGlu Met Val Thr Val Leu 20 25 30 Ser Ile Asp Gly Gly Gly Ile Arg Gly IleIle Pro Ala Thr Ile Leu 35 40 45 Glu Phe Leu Glu Gly Gln Leu Gln Glu MetAsp Asn Asn Ala Asp Ala 50 55 60 Arg Leu Ala Asp Tyr Phe Asp Val Ile GlyGly Thr Ser Thr Gly Gly 65 70 75 80 Leu Leu Thr Ala Met Ile Ser Thr ProAsn Glu Asn Asn Arg Pro Phe 85 90 95 Ala Ala Ala Lys Glu Ile Val Pro PheTyr Phe Glu His Gly Pro Gln 100 105 110 Ile Phe Asn Pro Ser Gly Gln IleLeu Gly Pro Lys Tyr Asp Gly Lys 115 120 125 Tyr Leu Met Gln Val Leu GlnGlu Lys Leu Gly Glu Thr Arg Val His 130 135 140 Gln Ala Leu Thr Glu ValVal Ile Ser Ser Phe Asp Ile Lys Thr Asn 145 150 155 160 Lys Pro Val IlePhe Thr Lys Ser Asn Leu Ala Asn Ser Pro Glu Leu 165 170 175 Asp Ala LysMet Tyr Asp Ile Ser Tyr Ser Thr Ala Ala Ala Pro Thr 180 185 190 Tyr PhePro Pro His Tyr Phe Val Thr Asn Thr Ser Asn Gly Asp Glu 195 200 205 TyrGlu Phe Asn Leu Val Asp Gly Ala Val Ala Thr Val Ala Asp Pro 210 215 220Ala Leu Leu Ser Ile Ser Val Ala Thr Arg Leu Ala Gln Lys Asp Pro 225 230235 240 Ala Phe Ala Ser Ile Arg Ser Leu Asn Tyr Lys Lys Met Leu Leu Leu245 250 255 Ser Leu Gly Thr Gly Thr Thr Ser Glu Phe Asp Lys Thr Tyr ThrAla 260 265 270 Lys Glu Ala Ala Thr Trp Thr Ala Val His Trp Met Leu ValIle Gln 275 280 285 Lys Met Thr Asp Ala Ala Ser Ser Tyr Met Thr Asp TyrTyr Leu Ser 290 295 300 Thr Ala Phe Gln Ala Leu Asp Ser Lys Asn Asn TyrLeu Arg Val Gln 305 310 315 320 Glu Asn Ala Leu Thr Gly Thr Thr Thr GluMet Asp Asp Ala Ser Glu 325 330 335 Ala Asn Met Glu Leu Leu Val Gln ValGly Glu Asn Leu Leu Lys Lys 340 345 350 Pro Val Ser Glu Asp Asn Pro GluThr Tyr Glu Glu Ala Leu Lys Arg 355 360 365 Phe Ala Lys Leu Leu Ser AspArg Lys Lys Leu Arg Ala Asn Lys Ala 370 375 380 Ser Tyr 385 2 365 PRTsynthetic Protein (1)..(365) Patatin isozyme PatFm (mature proteinlacking signal peptide) 2 Met Ala Leu Glu Glu Met Val Ala Val Leu SerIle Asp Gly Gly Gly 1 5 10 15 Ile Lys Gly Ile Ile Pro Gly Thr Ile LeuGlu Phe Leu Glu Gly Gln 20 25 30 Leu Gln Lys Met Asp Asn Asn Ala Asp AlaArg Leu Ala Asp Tyr Phe 35 40 45 Asp Val Ile Gly Gly Thr Ser Thr Gly GlyLeu Leu Thr Ala Met Ile 50 55 60 Thr Thr Pro Asn Glu Asn Asn Arg Pro PheAla Ala Ala Asn Glu Ile 65 70 75 80 Val Pro Phe Tyr Phe Glu His Gly ProHis Ile Phe Asn Ser Arg Tyr 85 90 95 Trp Pro Ile Phe Trp Pro Lys Tyr AspGly Lys Tyr Leu Met Gln Val 100 105 110 Leu Gln Glu Lys Leu Gly Glu ThrArg Val His Gln Ala Leu Thr Glu 115 120 125 Val Ala Ile Ser Ser Phe AspIle Lys Thr Asn Lys Pro Val Ile Phe 130 135 140 Thr Lys Ser Asn Leu AlaLys Ser Pro Glu Leu Asp Ala Lys Thr Tyr 145 150 155 160 Asp Ile Cys TyrSer Thr Ala Ala Ala Pro Thr Tyr Phe Pro Pro His 165 170 175 Tyr Phe AlaThr Asn Thr Ile Asn Gly Asp Lys Tyr Glu Phe Asn Leu 180 185 190 Val AspGly Ala Val Ala Thr Val Ala Asp Pro Ala Leu Leu Ser Val 195 200 205 SerVal Ala Thr Arg Arg Ala Gln Glu Asp Pro Ala Phe Ala Ser Ile 210 215 220Arg Ser Leu Asn Tyr Lys Lys Met Leu Leu Leu Ser Leu Gly Thr Gly 225 230235 240 Thr Thr Ser Glu Phe Asp Lys Thr His Thr Ala Glu Glu Thr Ala Lys245 250 255 Trp Gly Ala Leu Gln Trp Met Leu Val Ile Gln Gln Met Thr GluAla 260 265 270 Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser Thr Val PheGln Asp 275 280 285 Leu His Ser Gln Asn Asn Tyr Leu Arg Val Gln Glu AsnAla Leu Thr 290 295 300 Gly Thr Thr Thr Lys Ala Asp Asp Ala Ser Glu AlaAsn Met Glu Leu 305 310 315 320 Leu Ala Gln Val Gly Glu Asn Leu Leu LysLys Pro Val Ser Lys Asp 325 330 335 Asn Pro Glu Thr Tyr Glu Glu Ala LeuLys Arg Phe Ala Lys Leu Leu 340 345 350 Ser Asp Arg Lys Lys Leu Arg AlaAsn Lys Ala Ser Tyr 355 360 365 3 364 PRT synthetic Protein (1)..(364)Patatin isozyme PatIm (mature protein lacking signal peptide) 3 Pro TrpLeu Glu Glu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly 1 5 10 15 IleLys Gly Ile Ile Pro Ala Ile Ile Leu Glu Phe Leu Glu Gly Gln 20 25 30 LeuGln Glu Val Asp Asn Asn Lys Asp Ala Arg Leu Ala Asp Tyr Phe 35 40 45 AspVal Ile Gly Gly Thr Ser Thr Gly Gly Leu Leu Thr Ala Met Ile 50 55 60 ThrThr Pro Asn Glu Asn Asn Arg Pro Phe Ala Ala Ala Lys Asp Ile 65 70 75 80Val Pro Phe Tyr Phe Glu His Gly Pro His Ile Phe Asn Tyr Ser Gly 85 90 95Ser Ile Leu Gly Pro Met Tyr Asp Gly Lys Tyr Leu Leu Gln Val Leu 100 105110 Gln Glu Lys Leu Gly Glu Thr Arg Val His Gln Ala Leu Thr Glu Val 115120 125 Ala Ile Ser Ser Phe Asp Ile Lys Thr Asn Lys Pro Val Ile Phe Thr130 135 140 Lys Ser Asn Leu Ala Lys Ser Pro Glu Leu Asp Ala Lys Met TyrAsp 145 150 155 160 Ile Cys Tyr Ser Thr Ala Ala Ala Pro Ile Tyr Phe ProPro His His 165 170 175 Phe Val Thr His Thr Ser Asn Gly Ala Arg Tyr GluPhe Asn Leu Val 180 185 190 Asp Gly Ala Val Ala Thr Val Gly Asp Pro AlaLeu Leu Ser Leu Ser 195 200 205 Val Ala Thr Arg Leu Ala Gln Glu Asp ProAla Phe Ser Ser Ile Lys 210 215 220 Ser Leu Asp Tyr Lys Gln Met Leu LeuLeu Ser Leu Gly Thr Gly Thr 225 230 235 240 Asn Ser Glu Phe Asp Lys ThrTyr Thr Ala Glu Glu Ala Ala Lys Trp 245 250 255 Gly Pro Leu Arg Trp MetLeu Ala Ile Gln Gln Met Thr Asn Ala Ala 260 265 270 Ser Phe Tyr Met ThrAsp Tyr Tyr Ile Ser Thr Val Phe Gln Ala Arg 275 280 285 His Ser Gln AsnAsn Tyr Leu Arg Val Gln Glu Asn Ala Leu Asn Gly 290 295 300 Thr Thr ThrGlu Met Asp Asp Ala Ser Glu Ala Asn Met Glu Leu Leu 305 310 315 320 ValGln Val Gly Glu Thr Leu Leu Lys Lys Pro Val Ser Arg Asp Ser 325 330 335Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe Ala Lys Leu Leu Ser 340 345350 Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser Tyr 355 360 4 386 PRTsynthetic Protein (1)..(386) Patatin isozyme PatL+ (including signalpeptide) 4 Met Ala Thr Thr Lys Ser Phe Leu Ile Leu Phe Phe Met Ile LeuAla 1 5 10 15 Thr Thr Ser Ser Thr Cys Ala Lys Leu Glu Glu Met Val ThrVal Leu 20 25 30 Ser Ile Asp Gly Gly Gly Ile Lys Gly Ile Ile Pro Ala IleIle Leu 35 40 45 Glu Phe Leu Glu Gly Gln Leu Gln Glu Val Asp Asn Asn LysAsp Ala 50 55 60 Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser ThrGly Gly 65 70 75 80 Leu Leu Thr Ala Met Ile Thr Thr Pro Asn Glu Asn AsnArg Pro Phe 85 90 95 Ala Ala Ala Lys Asp Ile Val Pro Phe Tyr Phe Glu HisGly Pro His 100 105 110 Ile Phe Asn Tyr Ser Gly Ser Ile Leu Gly Pro MetTyr Asp Gly Lys 115 120 125 Tyr Leu Leu Gln Val Leu Gln Glu Lys Leu GlyGlu Thr Arg Val His 130 135 140 Gln Ala Leu Thr Glu Val Ala Ile Ser SerPhe Asp Ile Lys Thr Asn 145 150 155 160 Lys Pro Val Ile Phe Thr Lys SerAsn Leu Ala Lys Ser Pro Glu Leu 165 170 175 Asp Ala Lys Met Tyr Asp IleCys Tyr Ser Thr Ala Ala Ala Pro Ile 180 185 190 Tyr Phe Pro Pro His HisPhe Val Thr His Thr Ser Asn Gly Ala Arg 195 200 205 Tyr Glu Phe Asn LeuVal Asp Gly Ala Val Ala Thr Val Gly Asp Pro 210 215 220 Ala Leu Leu SerLeu Ser Val Ala Thr Arg Leu Ala Gln Glu Asp Pro 225 230 235 240 Ala PheSer Ser Ile Lys Ser Leu Asp Tyr Lys Gln Met Leu Leu Leu 245 250 255 SerLeu Gly Thr Gly Thr Asn Ser Glu Phe Asp Lys Thr Tyr Thr Ala 260 265 270Glu Glu Ala Ala Lys Trp Gly Pro Leu Arg Trp Met Leu Ala Ile Gln 275 280285 Gln Met Thr Asn Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Ile Ser 290295 300 Thr Val Phe Gln Ala Arg His Ser Gln Asn Asn Tyr Leu Arg Val Gln305 310 315 320 Glu Asn Ala Leu Asn Gly Thr Thr Thr Glu Met Asp Asp AlaSer Glu 325 330 335 Ala Asn Met Glu Leu Leu Val Gln Val Gly Ala Thr LeuLeu Lys Lys 340 345 350 Pro Val Ser Lys Asp Ser Pro Glu Thr Tyr Glu GluAla Leu Lys Arg 355 360 365 Phe Ala Lys Leu Leu Ser Asp Arg Lys Lys LeuArg Ala Asn Lys Ala 370 375 380 Ser Tyr 385 5 386 PRT synthetic Protein(1)..(386) Patatin isozyme PatA+ (including signal peptide) 5 Met AlaThr Thr Lys Ser Phe Leu Ile Leu Phe Phe Met Ile Leu Ala 1 5 10 15 ThrThr Ser Ser Thr Cys Ala Lys Leu Glu Glu Met Val Thr Val Leu 20 25 30 SerIle Asp Gly Gly Gly Ile Lys Gly Ile Ile Pro Ala Ile Ile Leu 35 40 45 GluPhe Leu Glu Gly Gln Leu Gln Glu Val Asp Asn Asn Lys Asp Ala 50 55 60 ArgLeu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly 65 70 75 80Leu Leu Thr Ala Met Ile Thr Thr Pro Asn Glu Asn Asn Arg Pro Phe 85 90 95Ala Ala Ala Lys Asp Ile Val Pro Phe Tyr Phe Glu His Gly Pro His 100 105110 Ile Phe Asn Tyr Ser Gly Ser Ile Ile Gly Pro Met Tyr Asp Gly Lys 115120 125 Tyr Leu Leu Gln Val Leu Gln Glu Lys Leu Gly Glu Thr Arg Val His130 135 140 Gln Ala Leu Thr Glu Val Ala Ile Ser Ser Phe Asp Ile Lys ThrAsn 145 150 155 160 Lys Pro Val Ile Phe Thr Lys Ser Asn Leu Ala Lys SerPro Glu Leu 165 170 175 Asp Ala Lys Met Tyr Asp Ile Cys Tyr Ser Thr AlaAla Ala Pro Ile 180 185 190 Tyr Phe Pro Pro His Tyr Phe Ile Thr His ThrSer Asn Gly Asp Ile 195 200 205 Tyr Glu Phe Asn Leu Val Asp Gly Gly ValAla Thr Val Gly Asp Pro 210 215 220 Ala Leu Leu Ser Leu Ser Val Ala ThrArg Leu Ala Gln Glu Asp Pro 225 230 235 240 Ala Phe Ser Ser Ile Lys SerLeu Asp Tyr Lys Gln Met Leu Leu Leu 245 250 255 Ser Leu Gly Thr Gly ThrAsn Ser Glu Phe Asp Lys Thr Tyr Thr Ala 260 265 270 Gln Glu Ala Ala LysTrp Gly Pro Leu Arg Trp Met Leu Ala Ile Gln 275 280 285 Gln Met Thr AsnAla Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Ile Ser 290 295 300 Thr Val PheGln Ala Arg His Ser Gln Asn Asn Tyr Leu Arg Val Gln 305 310 315 320 GluAsn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser Glu 325 330 335Ala Asn Met Glu Leu Leu Val Gln Val Gly Glu Thr Leu Leu Lys Lys 340 345350 Pro Val Ser Lys Asp Ser Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg 355360 365 Phe Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala370 375 380 Ser Tyr 385 6 386 PRT synthetic Protein (1)..(386) Patatinisozyme PatB+ (including signal peptide) 6 Met Ala Thr Thr Lys Ser ValLeu Val Leu Phe Phe Met Ile Leu Ala 1 5 10 15 Thr Thr Ser Ser Thr CysAla Thr Leu Gly Glu Met Val Thr Val Leu 20 25 30 Ser Ile Asp Gly Gly GlyIle Lys Gly Ile Ile Pro Ala Thr Ile Leu 35 40 45 Glu Phe Leu Glu Gly GlnLeu Gln Glu Val Asp Asn Asn Lys Asp Ala 50 55 60 Arg Leu Ala Asp Tyr PheAsp Val Ile Gly Gly Thr Ser Thr Gly Gly 65 70 75 80 Leu Leu Thr Ala MetIle Thr Thr Pro Asn Glu Asn Asn Arg Pro Phe 85 90 95 Ala Ala Ala Lys AspIle Val Pro Phe Tyr Phe Glu His Gly Pro His 100 105 110 Ile Phe Asn SerSer Gly Ser Ile Phe Gly Pro Met Tyr Asp Gly Lys 115 120 125 Tyr Phe LeuGln Val Leu Gln Glu Lys Leu Gly Glu Thr Arg Val His 130 135 140 Gln AlaLeu Thr Glu Val Ala Ile Ser Ser Phe Asp Ile Lys Thr Asn 145 150 155 160Lys Pro Val Ile Phe Thr Lys Ser Asn Leu Ala Lys Ser Pro Glu Leu 165 170175 Asp Ala Lys Met Asn Asp Ile Cys Tyr Ser Thr Ala Ala Ala Pro Thr 180185 190 Tyr Phe Pro Pro His Tyr Phe Val Thr His Thr Ser Asn Gly Asp Lys195 200 205 Tyr Glu Phe Asn Leu Val Asp Gly Ala Val Ala Thr Val Gly AspPro 210 215 220 Ala Leu Leu Ser Leu Ser Val Arg Thr Lys Leu Ala Gln ValAsp Pro 225 230 235 240 Lys Phe Ala Ser Ile Lys Ser Leu Asn Tyr Asn GluMet Leu Leu Leu 245 250 255 Ser Leu Gly Thr Gly Thr Asn Ser Glu Phe AspLys Thr Tyr Thr Ala 260 265 270 Glu Glu Ala Ala Lys Trp Gly Pro Leu ArgTrp Ile Leu Ala Ile Gln 275 280 285 Gln Met Thr Asn Ala Ala Ser Ser TyrMet Thr Asp Tyr Tyr Leu Ser 290 295 300 Thr Val Phe Gln Ala Arg His SerGln Asn Asn Tyr Leu Arg Val Gln 305 310 315 320 Glu Asn Ala Leu Thr GlyThr Thr Thr Glu Met Asp Asp Ala Ser Glu 325 330 335 Ala Asn Met Glu LeuLeu Val Gln Val Gly Glu Lys Leu Leu Lys Lys 340 345 350 Pro Val Ser LysAsp Ser Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg 355 360 365 Phe Ala LysLeu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala 370 375 380 Ser Tyr385 7 408 PRT Pentaclethra macroloba Protein (1)..(408) patatin homologpentin 1 7 Met Lys Ser Lys Met Ala Met Leu Leu Leu Leu Phe Cys Val LeuSer 1 5 10 15 Asn Gln Leu Val Ala Ala Phe Ser Thr Gln Ala Lys Ala SerLys Asp 20 25 30 Gly Asn Leu Val Thr Val Leu Ala Ile Asp Gly Gly Gly IleArg Gly 35 40 45 Ile Ile Pro Gly Val Ile Leu Lys Gln Leu Glu Ala Thr LeuGln Arg 50 55 60 Trp Asp Ser Ser Ala Arg Leu Ala Glu Tyr Phe Asp Val ValAla Gly 65 70 75 80 Thr Ser Thr Gly Gly Ile Ile Thr Ala Ile Leu Thr AlaPro Asp Pro 85 90 95 Gln Asn Lys Asp Arg Pro Leu Tyr Ala Ala Glu Glu IleIle Asp Phe 100 105 110 Tyr Ile Glu His Gly Pro Ser Ile Phe Asn Lys SerThr Ala Cys Ser 115 120 125 Leu Pro Gly Ile Phe Cys Pro Lys Tyr Asp GlyLys Tyr Leu Gln Glu 130 135 140 Ile Ile Ser Gln Lys Leu Asn Glu Thr LeuLeu Asp Gln Thr Thr Thr 145 150 155 160 Asn Val Val Ile Pro Ser Phe AspIle Lys Leu Leu Arg Pro Thr Ile 165 170 175 Phe Ser Thr Phe Lys Leu GluGlu Val Pro Glu Leu Asn Val Lys Leu 180 185 190 Ser Asp Val Cys Met GlyThr Ser Ala Ala Pro Ile Val Phe Pro Pro 195 200 205 Tyr Tyr Phe Lys HisGly Asp Thr Glu Phe Asn Leu Val Asp Gly Ala 210 215 220 Ile Ile Ala AspIle Pro Ala Pro Val Ala Leu Ser Glu Val Leu Gln 225 230 235 240 Gln GluLys Tyr Lys Asn Lys Glu Ile Leu Leu Leu Ser Ile Gly Thr 245 250 255 GlyVal Val Lys Pro Gly Glu Gly Tyr Ser Ala Asn Arg Thr Trp Thr 260 265 270Ile Phe Asp Trp Ser Ser Glu Thr Leu Ile Gly Leu Met Gly His Gly 275 280285 Thr Arg Ala Met Ser Asp Tyr Tyr Val Gly Ser His Phe Lys Ala Leu 290295 300 Gln Pro Gln Asn Asn Tyr Leu Arg Ile Gln Glu Tyr Asp Leu Asp Pro305 310 315 320 Ala Leu Glu Ser Ile Asp Asp Ala Ser Thr Glu Asn Met GluAsn Leu 325 330 335 Glu Lys Val Gly Gln Ser Leu Leu Asn Glu Pro Val LysArg Met Asn 340 345 350 Leu Asn Thr Phe Val Val Glu Glu Thr Gly Glu GlyThr Asn Ala Glu 355 360 365 Ala Leu Asp Arg Leu Ala Gln Ile Leu Tyr GluGlu Lys Ile Thr Arg 370 375 380 Gly Leu Gly Lys Ile Ser Leu Glu Val AspAsn Ile Asp Pro Tyr Thr 385 390 395 400 Glu Arg Val Arg Lys Leu Leu Phe405 8 410 PRT Zea mays Protein (1)..(410) monocot patatin homolog 5c9 8Met Gly Ser Ile Gly Arg Gly Thr Ala Asn Cys Ala Thr Val Pro Gln 1 5 1015 Pro Pro Pro Ser Thr Gly Lys Leu Ile Thr Ile Leu Ser Ile Asp Gly 20 2530 Gly Gly Ile Arg Gly Leu Ile Pro Ala Thr Ile Ile Ala Tyr Leu Glu 35 4045 Ala Lys Leu Gln Glu Leu Asp Gly Pro Asp Ala Arg Ile Ala Asp Tyr 50 5560 Phe Asp Val Ile Ala Gly Thr Ser Thr Gly Ala Leu Leu Ala Ser Met 65 7075 80 Leu Ala Ala Pro Asp Glu Asn Asn Arg Pro Leu Phe Ala Ala Lys Asp 8590 95 Leu Thr Thr Phe Tyr Leu Glu Asn Gly Pro Lys Ile Phe Pro Gln Lys100 105 110 Lys Ala Gly Leu Leu Thr Pro Leu Arg Asn Leu Leu Gly Leu ValArg 115 120 125 Gly Pro Lys Tyr Asp Gly Val Phe Leu His Asp Lys Ile LysSer Leu 130 135 140 Thr His Asp Val Arg Val Ala Asp Thr Val Thr Asn ValIle Val Pro 145 150 155 160 Ala Phe Asp Val Lys Tyr Leu Gln Pro Ile IlePhe Ser Thr Tyr Glu 165 170 175 Ala Lys Thr Asp Thr Leu Lys Asn Ala HisLeu Ser Asp Ile Cys Ile 180 185 190 Ser Thr Ser Ala Ala Pro Thr Tyr PhePro Ala His Phe Phe Lys Thr 195 200 205 Glu Ala Thr Asp Gly Arg Pro ProArg Glu Tyr His Leu Val Asp Gly 210 215 220 Gly Val Ala Ala Asn Asn ProThr Met Val Ala Met Ser Met Leu Thr 225 230 235 240 Lys Glu Val His ArgArg Asn Pro Asn Phe Asn Ala Gly Ser Pro Thr 245 250 255 Glu Tyr Thr AsnTyr Leu Ile Ile Ser Val Gly Thr Gly Ser Ala Lys 260 265 270 Gln Ala GluLys Tyr Thr Ala Glu Gln Cys Ala Lys Trp Gly Leu Ile 275 280 285 Gln TrpLeu Tyr Asn Gly Gly Phe Thr Pro Ile Ile Asp Ile Phe Ser 290 295 300 HisAla Ser Ser Asp Met Val Asp Ile His Ala Ser Ile Leu Phe Gln 305 310 315320 Ala Leu His Cys Glu Lys Lys Tyr Leu Arg Ile Gln Asp Asp Thr Leu 325330 335 Thr Gly Asn Ala Ser Ser Val Asp Ile Ala Thr Lys Glu Asn Met Glu340 345 350 Ser Leu Ile Ser Ile Gly Gln Glu Leu Leu Lys Lys Pro Val AlaArg 355 360 365 Val Asn Ile Asp Thr Gly Val Tyr Glu Ser Cys Asp Gly GluGly Thr 370 375 380 Asn Ala Gln Ser Leu Ala Asp Phe Ala Lys Gln Leu SerAsp Glu Arg 385 390 395 400 Lys Leu Arg Lys Ser Asn Leu Asn Ser Asn 405410 9 508 PRT synthetic Protein (1)..(508) maize patatin homolog aminoacid sequence corn 1 9 Arg Pro Thr Arg Pro Arg His Pro Arg Asn Thr GlnLys Arg Gly Ala 1 5 10 15 Leu Leu Val Gly Trp Ile Leu Phe Ser Leu AlaAla Ser Pro Val Lys 20 25 30 Phe Gln Thr His Met Gly Ser Ile Gly Arg GlyThr Ala Asn Cys Ala 35 40 45 Thr Val Pro Gln Pro Pro Pro Ser Thr Gly LysLeu Ile Thr Ile Leu 50 55 60 Ser Ile Asp Gly Gly Gly Ile Arg Gly Leu IlePro Ala Thr Ile Ile 65 70 75 80 Ala Tyr Leu Glu Ala Lys Leu Gln Glu LeuAsp Gly Pro Asp Ala Arg 85 90 95 Ile Ala Asp Tyr Phe Asp Val Ile Ala GlyThr Ser Thr Gly Ala Leu 100 105 110 Leu Ala Ser Met Leu Ala Ala Pro AspGlu Asn Asn Arg Pro Leu Phe 115 120 125 Ala Ala Lys Asp Leu Thr Thr PheTyr Leu Glu Asn Gly Pro Lys Ile 130 135 140 Phe Pro Gln Lys Lys Ala GlyLeu Leu Thr Pro Leu Arg Asn Leu Leu 145 150 155 160 Gly Leu Val Arg GlyPro Lys Tyr Asp Gly Val Phe Leu His Asp Lys 165 170 175 Ile Lys Ser LeuThr His Asp Val Arg Val Ala Asp Thr Val Thr Asn 180 185 190 Val Ile ValPro Ala Phe Asp Val Lys Tyr Leu Gln Pro Ile Ile Phe 195 200 205 Ser ThrTyr Glu Ala Lys Thr Asp Ala Leu Lys Asn Ala His Leu Ser 210 215 220 AspIle Cys Ile Ser Thr Ser Ala Ala Pro Thr Tyr Phe Pro Ala His 225 230 235240 Phe Phe Lys Thr Glu Ala Thr Asp Gly Arg Pro Pro Arg Glu Tyr His 245250 255 Leu Val Asp Gly Gly Val Ala Ala Asn Asn Pro Thr Met Val Ala Met260 265 270 Ser Met Leu Thr Lys Glu Val His Arg Arg Asn Pro Asn Phe AsnAla 275 280 285 Gly Ser Pro Thr Glu Tyr Thr Asn Tyr Leu Ile Ile Ser ValGly Thr 290 295 300 Gly Ser Ala Lys Gln Ala Glu Lys Tyr Thr Ala Glu GlnCys Ala Lys 305 310 315 320 Trp Gly Leu Ile Gln Trp Leu Tyr Asn Gly GlyPhe Thr Pro Ile Ile 325 330 335 Asp Ile Phe Ser His Ala Ser Ser Asp MetVal Asp Ile His Ala Ser 340 345 350 Ile Leu Phe Gln Ala Leu His Cys GluLys Lys Tyr Leu Arg Ile Gln 355 360 365 Leu Tyr Tyr Ala Gly Tyr Phe AspTrp Glu Arg Ile Val Arg Gly His 370 375 380 Arg His Gln Gly Glu His GlyVal Ser Asp Ile Asp Arg Pro Gly Ala 385 390 395 400 Ala Gln Glu Ala SerGly Glu Ser Glu His Arg His Arg Ala Val Arg 405 410 415 Val Leu Arg ArgGly His Lys Cys Thr Val Ala Ser Leu Arg Gln Ala 420 425 430 Thr Leu ArgAla Gln Ala Thr Gln Glu Gln Ser Gln Leu Gln Leu Ile 435 440 445 Asn ThrSer Leu Ser His Ser Met Cys Ser Phe Arg Arg Phe Thr Val 450 455 460 SerTyr Phe Phe Asn Phe Asn Ser Val Cys Val Leu Cys Val Leu Cys 465 470 475480 Val Tyr Gln Thr Phe Lys Phe Asn Gln Lys Lys Lys Lys Lys Lys Lys 485490 495 Lys Lys Lys Lys Lys Lys Lys Lys Lys Arg Ala Ala 500 505 10 410PRT synthetic Protein (1)..(410) maize patatin homolog amino acidsequence corn 2 10 Met Gly Ser Ile Gly Arg Gly Thr Ala Asn Cys Ala ThrVal Pro Gln 1 5 10 15 Pro Pro Pro Ser Thr Gly Lys Leu Ile Thr Ile LeuSer Ile Asp Gly 20 25 30 Gly Gly Ile Arg Gly Leu Ile Pro Ala Thr Ile IleAla Tyr Leu Glu 35 40 45 Ala Lys Leu Gln Glu Leu Asp Gly Pro Asp Ala ArgIle Ala Asp Tyr 50 55 60 Phe Asp Val Ile Ala Gly Thr Ser Thr Gly Ala LeuLeu Ala Ser Met 65 70 75 80 Leu Ala Ala Pro Asp Glu Asn Asn Arg Pro LeuPhe Ala Ala Lys Asp 85 90 95 Leu Thr Thr Phe Tyr Leu Glu Asn Gly Pro LysIle Phe Pro Gln Lys 100 105 110 Lys Ala Gly Leu Leu Thr Pro Leu Arg AsnLeu Leu Gly Leu Val Arg 115 120 125 Gly Pro Lys Tyr Asp Gly Val Phe LeuHis Asp Lys Ile Lys Ser Leu 130 135 140 Thr His Asp Val Arg Val Ala AspThr Val Thr Asn Val Ile Val Pro 145 150 155 160 Ala Phe Asp Val Lys SerLeu Gln Pro Ile Ile Phe Ser Thr Tyr Glu 165 170 175 Ala Lys Thr Asp ThrLeu Lys Asn Ala His Leu Ser Asp Ile Cys Ile 180 185 190 Ser Thr Ser AlaAla Pro Thr Tyr Phe Pro Ala His Phe Phe Lys Thr 195 200 205 Glu Ala ThrAsp Gly Arg Pro Pro Arg Glu Tyr His Leu Val Asp Gly 210 215 220 Gly ValAla Ala Asn Asn Pro Thr Met Val Ala Met Ser Met Leu Thr 225 230 235 240Lys Glu Val His Arg Arg Asn Pro Asn Phe Asn Ala Gly Ser Pro Thr 245 250255 Glu Tyr Thr Asn Tyr Leu Ile Ile Ser Val Gly Thr Gly Ser Ala Lys 260265 270 Gln Ala Glu Lys Tyr Thr Ala Glu Gln Cys Ala Lys Trp Gly Leu Ile275 280 285 Gln Trp Leu Tyr Asn Gly Gly Phe Thr Pro Ile Ile Asp Ile PheSer 290 295 300 His Ala Ser Ser Asp Met Val Asp Ile His Ala Ser Ile LeuPhe Gln 305 310 315 320 Ala Leu His Cys Glu Lys Lys Tyr Leu Arg Ile GlnAsp Asp Thr Leu 325 330 335 Thr Gly Asn Ala Ser Ser Val Asp Ile Ala ThrLys Glu Asn Met Glu 340 345 350 Ser Leu Ile Ser Ile Gly Gln Glu Leu LeuAsn Lys Pro Val Ala Arg 355 360 365 Val Asn Ile Asp Thr Gly Leu Tyr GluSer Cys Glu Gly Glu Gly Thr 370 375 380 Asn Ala Gln Ser Leu Ala Asp PheAla Lys Gln Leu Ser Asp Glu Arg 385 390 395 400 Lys Leu Arg Lys Ser AsnLeu Asn Ser Asn 405 410 11 410 PRT synthetic Protein (1)..(410) maizepatatin homolog amino acid sequence corn 3 11 Met Gly Ser Ile Gly ArgGly Thr Ala Asn Cys Ala Thr Val Pro Gln 1 5 10 15 Pro Pro Pro Ser ThrGly Lys Leu Ile Thr Ile Leu Ser Ile Asp Gly 20 25 30 Gly Gly Ile Arg GlyLeu Ile Pro Ala Thr Ile Ile Ala Tyr Leu Glu 35 40 45 Ala Lys Leu Gln GluLeu Asp Gly Pro Asp Ala Arg Ile Ala Asp Tyr 50 55 60 Phe Asp Val Ile AlaGly Thr Ser Thr Gly Ala Leu Leu Ala Ser Met 65 70 75 80 Leu Ala Ala ProAsp Glu Asn Asn Arg Pro Leu Phe Ala Ala Lys Asp 85 90 95 Leu Thr Thr PheTyr Leu Glu Asn Gly Pro Lys Ile Phe Pro Gln Lys 100 105 110 Lys Ala GlyLeu Leu Thr Pro Leu Arg Asn Leu Leu Gly Leu Val Arg 115 120 125 Gly ProLys Tyr Asp Gly Val Phe Leu His Asp Lys Ile Lys Ser Leu 130 135 140 ThrHis Asp Val Arg Val Ala Asp Thr Val Thr Asn Val Ile Val Pro 145 150 155160 Ala Phe Asp Val Lys Tyr Leu Gln Pro Ile Ile Phe Ser Thr Tyr Glu 165170 175 Ala Lys Thr Asp Ala Leu Lys Asn Ala His Leu Ser Asp Ile Cys Ile180 185 190 Ser Thr Ser Ala Ala Pro Thr Tyr Phe Pro Ala His Phe Phe LysThr 195 200 205 Glu Ala Thr Asp Gly Arg Pro Pro Arg Glu Tyr His Leu ValAsp Gly 210 215 220 Gly Val Ala Ala Asn Asn Pro Thr Met Val Ala Met SerMet Leu Thr 225 230 235 240 Lys Glu Val His Arg Arg Asn Pro Asn Phe AsnAla Gly Ser Pro Thr 245 250 255 Glu Tyr Thr Asn Tyr Leu Ile Ile Ser ValGly Thr Gly Ser Ala Lys 260 265 270 Gln Ala Glu Lys Tyr Thr Ala Glu GlnCys Ala Lys Trp Gly Leu Ile 275 280 285 Gln Trp Leu Tyr Asn Gly Gly PheThr Pro Ile Ile Asp Ile Phe Ser 290 295 300 His Ala Ser Ser Asp Met ValAsp Ile His Ala Ser Ile Leu Phe Gln 305 310 315 320 Ala Leu His Cys GluLys Lys Tyr Leu Arg Ile Gln Asp Asp Thr Leu 325 330 335 Thr Gly Asn AlaSer Ser Val Asp Ile Ala Thr Lys Glu Asn Met Glu 340 345 350 Ser Leu IleSer Ile Gly Gln Glu Leu Leu Lys Lys Pro Val Ala Arg 355 360 365 Val AsnIle Asp Thr Gly Leu Tyr Glu Ser Cys Asp Gly Glu Gly Thr 370 375 380 AsnAla Gln Ser Leu Ala Asp Phe Ala Lys Gln Leu Ser Asp Glu Arg 385 390 395400 Lys Leu Arg Lys Ser Asn Leu Asn Ser Asn 405 410 12 410 PRT syntheticProtein (1)..(410) maize patatin homolog amino acid sequence corn 4 12Met Gly Ser Ile Gly Arg Gly Thr Ala Asn Cys Ala Thr Val Pro Gln 1 5 1015 Pro Pro Pro Ser Thr Gly Lys Leu Ile Thr Ile Leu Ser Ile Asp Gly 20 2530 Gly Gly Ile Arg Gly Leu Ile Pro Ala Thr Ile Ile Ala Tyr Leu Glu 35 4045 Ala Lys Leu Gln Glu Leu Asp Gly Pro Asp Ala Arg Ile Ala Asp Tyr 50 5560 Phe Asp Val Ile Ala Gly Thr Ser Thr Gly Ala Leu Leu Ala Ser Met 65 7075 80 Leu Ala Ala Pro Asp Glu Asn Asn Arg Pro Leu Phe Ala Ala Lys Asp 8590 95 Leu Thr Thr Phe Tyr Leu Glu Asn Gly Pro Lys Ile Phe Pro Gln Lys100 105 110 Lys Ala Gly Leu Leu Thr Pro Leu Arg Asn Leu Leu Gly Leu ValArg 115 120 125 Gly Pro Lys Tyr Asp Gly Val Phe Leu His Asp Lys Ile LysSer Leu 130 135 140 Thr His Asp Val Arg Val Ala Asp Thr Val Thr Asn ValIle Val Pro 145 150 155 160 Ala Phe Asp Val Lys Ser Leu Gln Pro Ile IlePhe Ser Thr Tyr Glu 165 170 175 Ala Lys Thr Asp Thr Leu Lys Asn Ala HisLeu Ser Asp Ile Cys Ile 180 185 190 Ser Thr Ser Ala Ala Pro Thr Tyr PhePro Ala His Phe Phe Lys Ile 195 200 205 Glu Ala Thr Asp Gly Arg Pro ProArg Glu Tyr His Leu Val Asp Gly 210 215 220 Gly Val Ala Ala Asn Asn ProThr Met Val Ala Met Ser Met Leu Thr 225 230 235 240 Lys Glu Val His ArgArg Asn Pro Asn Phe Asn Ala Gly Ser Pro Thr 245 250 255 Glu Tyr Thr AsnTyr Leu Ile Ile Ser Val Gly Thr Gly Ser Ala Lys 260 265 270 Gln Ala GluLys Tyr Thr Ala Glu Gln Cys Ala Lys Trp Gly Leu Ile 275 280 285 Gln TrpLeu Tyr Asn Gly Gly Phe Thr Pro Ile Ile Asp Ile Phe Ser 290 295 300 HisAla Ser Ser Asp Met Val Asp Ile His Ala Ser Ile Leu Phe Gln 305 310 315320 Ala Leu His Cys Glu Lys Lys Tyr Leu Arg Ile Gln Asp Asp Thr Leu 325330 335 Thr Gly Asn Ala Ser Ser Val Asp Ile Ala Thr Lys Glu Asn Met Glu340 345 350 Ser Leu Ile Ser Ile Gly Gln Glu Leu Leu Asn Lys Pro Val AlaArg 355 360 365 Val Asn Ile Asp Thr Gly Leu Tyr Glu Ser Cys Glu Gly GluGly Thr 370 375 380 Asn Ala Gln Ser Leu Ala Asp Phe Ala Lys Gln Leu SerAsp Glu Arg 385 390 395 400 Lys Leu Arg Lys Ser Asn Leu Asn Ser Asn 405410 13 337 PRT synthetic Protein (1)..(337) maize patatin homolog aminoacid sequence corn 5 13 Met Gly Ser Ile Gly Arg Gly Thr Ala Asn Cys AlaThr Val Pro Gln 1 5 10 15 Pro Pro Pro Ser Thr Gly Lys Leu Ile Thr IleLeu Ser Ile Asp Gly 20 25 30 Gly Gly Ile Arg Gly Leu Ile Pro Ala Thr IleIle Ala Tyr Leu Glu 35 40 45 Ala Lys Leu Gln Glu Leu Asp Gly Pro Asp AlaArg Ile Ala Asp Tyr 50 55 60 Phe Asp Val Ile Ala Gly Thr Ser Thr Gly AlaLeu Leu Ala Ser Met 65 70 75 80 Leu Ala Ala Pro Asp Glu Asn Asn Arg ProLeu Phe Ala Ala Lys Asp 85 90 95 Leu Thr Thr Phe Tyr Leu Glu Asn Gly ProLys Ile Phe Pro Gln Lys 100 105 110 Lys Ala Gly Leu Leu Thr Pro Leu ArgAsn Leu Leu Gly Leu Val Arg 115 120 125 Gly Pro Lys Tyr Asp Gly Val PheLeu His Asp Lys Ile Lys Ser Leu 130 135 140 Thr His Asp Val Arg Val AlaAsp Thr Val Thr Asn Val Ile Val Pro 145 150 155 160 Ala Phe Asp Val LysTyr Leu Gln Pro Ile Ile Phe Ser Thr Tyr Glu 165 170 175 Ala Lys Thr AspAla Leu Lys Asn Ala His Leu Ser Asp Ile Cys Ile 180 185 190 Ser Thr SerAla Ala Pro Thr Tyr Phe Pro Ala His Phe Phe Lys Thr 195 200 205 Glu AlaThr Asp Gly Arg Pro Pro Arg Glu Tyr His Leu Val Asp Gly 210 215 220 GlyVal Ala Ala Asn Asn Pro Thr Met Val Ala Met Ser Met Leu Thr 225 230 235240 Lys Glu Val His Arg Arg Asn Pro Asn Phe Asn Ala Gly Ser Pro Thr 245250 255 Glu Tyr Thr Asn Tyr Leu Ile Ile Ser Val Gly Thr Gly Ser Ala Lys260 265 270 Gln Ala Glu Lys Tyr Thr Ala Glu Gln Cys Ala Lys Trp Gly LeuIle 275 280 285 Gln Trp Leu Tyr Asn Gly Gly Phe Thr Pro Ile Ile Asp IlePhe Ser 290 295 300 His Ala Ser Ser Asp Met Val Asp Ile His Ala Ser IleLeu Phe Gln 305 310 315 320 Ala Leu His Cys Glu Lys Lys Tyr Leu Arg IleGln Leu Tyr Tyr Ala 325 330 335 Gly 14 5 PRT synthetic Protein (1)..(5)Xaa = Ser or Thr. 14 Gly Xaa Ser Xaa Gly 1 5 15 7 PRT synthetic Protein(1)..(7) Xaa2 = Aromatics such as Phe, Tyr, Trp. Xaa3 = Arg or His. 15Glu Xaa Xaa Leu Val Asp Gly 1 5 16 3 PRT synthetic Protein (1)..(3)Linker Sequence 16 Gly Pro Gly 1 17 7 PRT synthetic Protein (1)..(7)Linker Sequence 2 17 Gly Gly Gly Ser Gly Gly Gly 1 5 18 33 DNA syntheticDNA (1)..(33) oligonucleotide-1 18 gttagatctc accatggcaa ctactaaatc ttt33 19 33 DNA synthetic DNA (1)..(33) oligonucleotide-2 19 ccagaattctcattaataag aagctttgtt tgc 33 20 1128 DNA synthetic DNA (1)..(1128)pMON37402 sequence encoding permutein protein 20 tcgagaaaag agaggctgaagcttcattga attacaaaaa aatgctgttg ctctcattag 60 gcactggcac tacttcagagtttgataaaa catatacagc aaaagaggca gctacctgga 120 ctgctgtaca ttggatgttagttatacaga aaatgactga tgcagcaagt tcttacatga 180 ctgattatta cctttctactgcttttcaag ctcttgattc aaaaaacaat tacctcaggg 240 ttcaagaaaa tgcattaacaggcacaacta ctgaaatgga tgatgcttct gaggctaata 300 tggaattatt agtacaagttggtgaaaact tattgaagaa accagtttcc gaagacaatc 360 ctgaaaccta tgaggaagctctaaagaggt ttgcaaaatt gctctctgat aggaagaaac 420 tccgagcaaa caaagcttcttatggaccag gacagttggg agaaatggtg actgttctta 480 gtattgatgg aggtggaattagagggatca ttccggctac cattctcgaa tttcttgaag 540 gacaacttca ggaaatggacaataatgcag atgcaagact tgcagattac tttgatgtaa 600 ttggaggaac aagtacaggaggtttattga ctgctatgat aagtactcca aatgaaaaca 660 atcgaccctt tgctgctgccaaagaaattg taccttttta cttcgaacat ggccctcaga 720 tttttaatcc tagtggtcaaattttaggcc caaaatatga tggaaaatat cttatgcaag 780 ttcttcaaga aaaacttggagaaactcgtg tgcatcaagc tttgacagaa gttgtcatct 840 caagctttga catcaaaacaaataagccag taatattcac taagtcaaat ttagcaaact 900 ctccagaatt ggatgctaagatgtatgaca taagttattc cacagcagca gctccaacat 960 attttcctcc gcattactttgttactaata ctagtaatgg agatgaatat gagttcaatc 1020 ttgttgatgg tgctgttgctactgttgctg atccggcgtt attatccatt agcgttgcaa 1080 cgagacttgc acaaaaggatccagcatttg cttcaattag gtaatgag 1128 21 366 PRT synthetic Protein(1)..(366) Permutein protein encoded from pMON37402 sequence 21 Ser LeuAsn Tyr Lys Lys Met Leu Leu Leu Ser Leu Gly Thr Gly Thr 1 5 10 15 ThrSer Glu Phe Asp Lys Thr Tyr Thr Ala Lys Glu Ala Ala Thr Trp 20 25 30 ThrAla Val His Trp Met Leu Val Ile Gln Lys Met Thr Asp Ala Ala 35 40 45 SerSer Tyr Met Thr Asp Tyr Tyr Leu Ser Thr Ala Phe Gln Ala Leu 50 55 60 AspSer Lys Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala Leu Thr Gly 65 70 75 80Thr Thr Thr Glu Met Asp Asp Ala Ser Glu Ala Asn Met Glu Leu Leu 85 90 95Val Gln Val Gly Glu Asn Leu Leu Lys Lys Pro Val Ser Glu Asp Asn 100 105110 Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe Ala Lys Leu Leu Ser 115120 125 Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser Tyr Gly Pro Gly Gln130 135 140 Leu Gly Glu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly IleArg 145 150 155 160 Gly Ile Ile Pro Ala Thr Ile Leu Glu Phe Leu Glu GlyGln Leu Gln 165 170 175 Glu Met Asp Asn Asn Ala Asp Ala Arg Leu Ala AspTyr Phe Asp Val 180 185 190 Ile Gly Gly Thr Ser Thr Gly Gly Leu Leu ThrAla Met Ile Ser Thr 195 200 205 Pro Asn Glu Asn Asn Arg Pro Phe Ala AlaAla Lys Glu Ile Val Pro 210 215 220 Phe Tyr Phe Glu His Gly Pro Gln IlePhe Asn Pro Ser Gly Gln Ile 225 230 235 240 Leu Gly Pro Lys Tyr Asp GlyLys Tyr Leu Met Gln Val Leu Gln Glu 245 250 255 Lys Leu Gly Glu Thr ArgVal His Gln Ala Leu Thr Glu Val Val Ile 260 265 270 Ser Ser Phe Asp IleLys Thr Asn Lys Pro Val Ile Phe Thr Lys Ser 275 280 285 Asn Leu Ala AsnSer Pro Glu Leu Asp Ala Lys Met Tyr Asp Ile Ser 290 295 300 Tyr Ser ThrAla Ala Ala Pro Thr Tyr Phe Pro Pro His Tyr Phe Val 305 310 315 320 ThrAsn Thr Ser Asn Gly Asp Glu Tyr Glu Phe Asn Leu Val Asp Gly 325 330 335Ala Val Ala Thr Val Ala Asp Pro Ala Leu Leu Ser Ile Ser Val Ala 340 345350 Thr Arg Leu Ala Gln Lys Asp Pro Ala Phe Ala Ser Ile Arg 355 360 36522 1128 DNA synthetic DNA (1)..(1128) pMON37405 sequence encodingpermutein protein 22 tcgagaaaag agaggctgaa gctaatacta gtaatggagatgaatatgag ttcaatcttg 60 ttgatggtgc tgttgctact gttgctgatc cggcgttattatccattagc gttgcaacga 120 gacttgcaca aaaggatcca gcatttgctt caattaggtcattgaattac aaaaaaatgc 180 tgttgctctc attaggcact ggcactactt cagagtttgataaaacatat acagcaaaag 240 aggcagctac ctggactgct gtacattgga tgttagttatacagaaaatg actgatgcag 300 caagttctta catgactgat tattaccttt ctactgcttttcaagctctt gattcaaaaa 360 acaattacct cagggttcaa gaaaatgcat taacaggcacaactactgaa atggatgatg 420 cttctgaggc taatatggaa ttattagtac aagttggtgaaaacttattg aagaaaccag 480 tttccgaaga caatcctgaa acctatgagg aagctctaaagaggtttgca aaattgctct 540 ctgataggaa gaaactccga gcaaacaaag cttcttatggaccaggacag ttgggagaaa 600 tggtgactgt tcttagtatt gatggaggtg gaattagagggatcattccg gctaccattc 660 tcgaatttct tgaaggacaa cttcaggaaa tggacaataatgcagatgca agacttgcag 720 attactttga tgtaattgga ggaacaagta caggaggtttattgactgct atgataagta 780 ctccaaatga aaacaatcga ccctttgctg ctgccaaagaaattgtacct ttttacttcg 840 aacatggccc tcagattttt aatcctagtg gtcaaattttaggcccaaaa tatgatggaa 900 aatatcttat gcaagttctt caagaaaaac ttggagaaactcgtgtgcat caagctttga 960 cagaagttgt catctcaagc tttgacatca aaacaaataagccagtaata ttcactaagt 1020 caaatttagc aaactctcca gaattggatg ctaagatgtatgacataagt tattccacag 1080 cagcagctcc aacatatttt cctccgcatt actttgttacttaatgag 1128 23 366 PRT synthetic Protein (1)..(366) Permutein proteinencoded by pMON37405 sequence 23 Asn Thr Ser Asn Gly Asp Glu Tyr Glu PheAsn Leu Val Asp Gly Ala 1 5 10 15 Val Ala Thr Val Ala Asp Pro Ala LeuLeu Ser Ile Ser Val Ala Thr 20 25 30 Arg Leu Ala Gln Lys Asp Pro Ala PheAla Ser Ile Arg Ser Leu Asn 35 40 45 Tyr Lys Lys Met Leu Leu Leu Ser LeuGly Thr Gly Thr Thr Ser Glu 50 55 60 Phe Asp Lys Thr Tyr Thr Ala Lys GluAla Ala Thr Trp Thr Ala Val 65 70 75 80 His Trp Met Leu Val Ile Gln LysMet Thr Asp Ala Ala Ser Ser Tyr 85 90 95 Met Thr Asp Tyr Tyr Leu Ser ThrAla Phe Gln Ala Leu Asp Ser Lys 100 105 110 Asn Asn Tyr Leu Arg Val GlnGlu Asn Ala Leu Thr Gly Thr Thr Thr 115 120 125 Glu Met Asp Asp Ala SerGlu Ala Asn Met Glu Leu Leu Val Gln Val 130 135 140 Gly Glu Asn Leu LeuLys Lys Pro Val Ser Glu Asp Asn Pro Glu Thr 145 150 155 160 Tyr Glu GluAla Leu Lys Arg Phe Ala Lys Leu Leu Ser Asp Arg Lys 165 170 175 Lys LeuArg Ala Asn Lys Ala Ser Tyr Gly Pro Gly Gln Leu Gly Glu 180 185 190 MetVal Thr Val Leu Ser Ile Asp Gly Gly Gly Ile Arg Gly Ile Ile 195 200 205Pro Ala Thr Ile Leu Glu Phe Leu Glu Gly Gln Leu Gln Glu Met Asp 210 215220 Asn Asn Ala Asp Ala Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly 225230 235 240 Thr Ser Thr Gly Gly Leu Leu Thr Ala Met Ile Ser Thr Pro AsnGlu 245 250 255 Asn Asn Arg Pro Phe Ala Ala Ala Lys Glu Ile Val Pro PheTyr Phe 260 265 270 Glu His Gly Pro Gln Ile Phe Asn Pro Ser Gly Gln IleLeu Gly Pro 275 280 285 Lys Tyr Asp Gly Lys Tyr Leu Met Gln Val Leu GlnGlu Lys Leu Gly 290 295 300 Glu Thr Arg Val His Gln Ala Leu Thr Glu ValVal Ile Ser Ser Phe 305 310 315 320 Asp Ile Lys Thr Asn Lys Pro Val IlePhe Thr Lys Ser Asn Leu Ala 325 330 335 Asn Ser Pro Glu Leu Asp Ala LysMet Tyr Asp Ile Ser Tyr Ser Thr 340 345 350 Ala Ala Ala Pro Thr Tyr PhePro Pro His Tyr Phe Val Thr 355 360 365 24 1128 DNA synthetic DNA(1)..(1128) pMON37406 sequence encoding permutein protein 24 tcgagaaaagagaggctgaa gctagttatt ccacagcagc agctccaaca tattttcctc 60 cgcattactttgttactaat actagtaatg gagatgaata tgagttcaat cttgttgatg 120 gtgctgttgctactgttgct gatccggcgt tattatccat tagcgttgca acgagacttg 180 cacaaaaggatccagcattt gcttcaatta ggtcattgaa ttacaaaaaa atgctgttgc 240 tctcattaggcactggcact acttcagagt ttgataaaac atatacagca aaagaggcag 300 ctacctggactgctgtacat tggatgttag ttatacagaa aatgactgat gcagcaagtt 360 cttacatgactgattattac ctttctactg cttttcaagc tcttgattca aaaaacaatt 420 acctcagggttcaagaaaat gcattaacag gcacaactac tgaaatggat gatgcttctg 480 aggctaatatggaattatta gtacaagttg gtgaaaactt attgaagaaa ccagtttccg 540 aagacaatcctgaaacctat gaggaagctc taaagaggtt tgcaaaattg ctctctgata 600 ggaagaaactccgagcaaac aaagcttctt atggaccagg acagttggga gaaatggtga 660 ctgttcttagtattgatgga ggtggaatta gagggatcat tccggctacc attctcgaat 720 ttcttgaaggacaacttcag gaaatggaca ataatgcaga tgcaagactt gcagattact 780 ttgatgtaattggaggaaca agtacaggag gtttattgac tgctatgata agtactccaa 840 atgaaaacaatcgacccttt gctgctgcca aagaaattgt acctttttac ttcgaacatg 900 gccctcagatttttaatcct agtggtcaaa ttttaggccc aaaatatgat ggaaaatatc 960 ttatgcaagttcttcaagaa aaacttggag aaactcgtgt gcatcaagct ttgacagaag 1020 ttgtcatctcaagctttgac atcaaaacaa ataagccagt aatattcact aagtcaaatt 1080 tagcaaactctccagaattg gatgctaaga tgtatgacat ataatgag 1128 25 366 PRT syntheticProtein (1)..(366) Permutein protein encoded by pMON37406 25 Ser Tyr SerThr Ala Ala Ala Pro Thr Tyr Phe Pro Pro His Tyr Phe 1 5 10 15 Val ThrAsn Thr Ser Asn Gly Asp Glu Tyr Glu Phe Asn Leu Val Asp 20 25 30 Gly AlaVal Ala Thr Val Ala Asp Pro Ala Leu Leu Ser Ile Ser Val 35 40 45 Ala ThrArg Leu Ala Gln Lys Asp Pro Ala Phe Ala Ser Ile Arg Ser 50 55 60 Leu AsnTyr Lys Lys Met Leu Leu Leu Ser Leu Gly Thr Gly Thr Thr 65 70 75 80 SerGlu Phe Asp Lys Thr Tyr Thr Ala Lys Glu Ala Ala Thr Trp Thr 85 90 95 AlaVal His Trp Met Leu Val Ile Gln Lys Met Thr Asp Ala Ala Ser 100 105 110Ser Tyr Met Thr Asp Tyr Tyr Leu Ser Thr Ala Phe Gln Ala Leu Asp 115 120125 Ser Lys Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala Leu Thr Gly Thr 130135 140 Thr Thr Glu Met Asp Asp Ala Ser Glu Ala Asn Met Glu Leu Leu Val145 150 155 160 Gln Val Gly Glu Asn Leu Leu Lys Lys Pro Val Ser Glu AspAsn Pro 165 170 175 Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe Ala Lys LeuLeu Ser Asp 180 185 190 Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser Tyr GlyPro Gly Gln Leu 195 200 205 Gly Glu Met Val Thr Val Leu Ser Ile Asp GlyGly Gly Ile Arg Gly 210 215 220 Ile Ile Pro Ala Thr Ile Leu Glu Phe LeuGlu Gly Gln Leu Gln Glu 225 230 235 240 Met Asp Asn Asn Ala Asp Ala ArgLeu Ala Asp Tyr Phe Asp Val Ile 245 250 255 Gly Gly Thr Ser Thr Gly GlyLeu Leu Thr Ala Met Ile Ser Thr Pro 260 265 270 Asn Glu Asn Asn Arg ProPhe Ala Ala Ala Lys Glu Ile Val Pro Phe 275 280 285 Tyr Phe Glu His GlyPro Gln Ile Phe Asn Pro Ser Gly Gln Ile Leu 290 295 300 Gly Pro Lys TyrAsp Gly Lys Tyr Leu Met Gln Val Leu Gln Glu Lys 305 310 315 320 Leu GlyGlu Thr Arg Val His Gln Ala Leu Thr Glu Val Val Ile Ser 325 330 335 SerPhe Asp Ile Lys Thr Asn Lys Pro Val Ile Phe Thr Lys Ser Asn 340 345 350Leu Ala Asn Ser Pro Glu Leu Asp Ala Lys Met Tyr Asp Ile 355 360 365 261128 DNA synthetic DNA (1)..(1128) pMON37407 sequence encoding permuteinprotein 26 tcgagaaaag agaggctgaa gctacatata cagcaaaaga ggcagctacctggactgctg 60 tacattggat gttagttata cagaaaatga ctgatgcagc aagttcttacatgactgatt 120 attacctttc tactgctttt caagctcttg attcaaaaaa caattacctcagggttcaag 180 aaaatgcatt aacaggcaca actactgaaa tggatgatgc ttctgaggctaatatggaat 240 tattagtaca agttggtgaa aacttattga agaaaccagt ttccgaagacaatcctgaaa 300 cctatgagga agctctaaag aggtttgcaa aattgctctc tgataggaagaaactccgat 360 caaacaaagc ttcttatgga ccaggacagt tgggagaaat ggtgactgttcttagtattg 420 atggaggtgg aattagaggg atcattccgg ctaccattct cgaatttcttgaaggacaac 480 ttcaggaaat ggacaataat gcagatgcaa gacttgcaga ttactttgatgtaattggag 540 gaacaagtac aggaggttta ttgactgcta tgataagtac tccaaatgaaaacaatcgac 600 cctttgctgc tgccaaagaa attgtacctt tttacttcga acatggccctcagattttta 660 atcctagtgg tcaaatttta ggcccaaaat atgatggaaa atatcttatgcaagttcttc 720 aagaaaaact tggagaaact cgtgtgcatc aagctttgac agaagttgtcatctcaagct 780 ttgacatcaa aacaaataag ccagtaatat tcactaagtc aaatttagcaaactctccag 840 aattggatgc taagatgtat gacataagtt attccacagc agcagctccaacatattttc 900 ctccgcatta ctttgttact aatactagta atggagatga atatgagttcaatcttgttg 960 atggtgctgt tgctactgtt gctgatccgg cgttattatc cattagcgttgcaacgagac 1020 ttgcacaaaa ggatccagca tttgcttcaa ttaggtcatt gaattacaaaaaaatgctgt 1080 tgctctcatt aggcactggc actacttcag agtttgataa ataatgag1128 27 366 PRT synthetic Protein (1)..(366) Permutein protein encodedby pMON37407 sequence 27 Thr Tyr Thr Ala Lys Glu Ala Ala Thr Trp Thr AlaVal His Trp Met 1 5 10 15 Leu Val Ile Gln Lys Met Thr Asp Ala Ala SerSer Tyr Met Thr Asp 20 25 30 Tyr Tyr Leu Ser Thr Ala Phe Gln Ala Leu AspSer Lys Asn Asn Tyr 35 40 45 Leu Arg Val Gln Glu Asn Ala Leu Thr Gly ThrThr Thr Glu Met Asp 50 55 60 Asp Ala Ser Glu Ala Asn Met Glu Leu Leu ValGln Val Gly Glu Asn 65 70 75 80 Leu Leu Lys Lys Pro Val Ser Glu Asp AsnPro Glu Thr Tyr Glu Glu 85 90 95 Ala Leu Lys Arg Phe Ala Lys Leu Leu SerAsp Arg Lys Lys Leu Arg 100 105 110 Ser Asn Lys Ala Ser Tyr Gly Pro GlyGln Leu Gly Glu Met Val Thr 115 120 125 Val Leu Ser Ile Asp Gly Gly GlyIle Arg Gly Ile Ile Pro Ala Thr 130 135 140 Ile Leu Glu Phe Leu Glu GlyGln Leu Gln Glu Met Asp Asn Asn Ala 145 150 155 160 Asp Ala Arg Leu AlaAsp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr 165 170 175 Gly Gly Leu LeuThr Ala Met Ile Ser Thr Pro Asn Glu Asn Asn Arg 180 185 190 Pro Phe AlaAla Ala Lys Glu Ile Val Pro Phe Tyr Phe Glu His Gly 195 200 205 Pro GlnIle Phe Asn Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp 210 215 220 GlyLys Tyr Leu Met Gln Val Leu Gln Glu Lys Leu Gly Glu Thr Arg 225 230 235240 Val His Gln Ala Leu Thr Glu Val Val Ile Ser Ser Phe Asp Ile Lys 245250 255 Thr Asn Lys Pro Val Ile Phe Thr Lys Ser Asn Leu Ala Asn Ser Pro260 265 270 Glu Leu Asp Ala Lys Met Tyr Asp Ile Ser Tyr Ser Thr Ala AlaAla 275 280 285 Pro Thr Tyr Phe Pro Pro His Tyr Phe Val Thr Asn Thr SerAsn Gly 290 295 300 Asp Glu Tyr Glu Phe Asn Leu Val Asp Gly Ala Val AlaThr Val Ala 305 310 315 320 Asp Pro Ala Leu Leu Ser Ile Ser Val Ala ThrArg Leu Ala Gln Lys 325 330 335 Asp Pro Ala Phe Ala Ser Ile Arg Ser LeuAsn Tyr Lys Lys Met Leu 340 345 350 Leu Leu Ser Leu Gly Thr Gly Thr ThrSer Glu Phe Asp Lys 355 360 365 28 1128 DNA synthetic DNA (1)..(1128)pMON37408 sequence encoding permutein protein 28 tcgagaaaag agaggctgaagctaatgcat taacaggcac aactactgaa atggatgatg 60 cttctgaggc taatatggaattattagtac aagttggtga aaacttattg aagaaaccag 120 tttccgaaga caatcctgaaacctatgagg aagctctaaa gaggtttgca aaattgctct 180 ctgataggaa gaaactccgagcaaacaaag cttcttatgg accaggacag ttgggagaaa 240 tggtgactgt tcttagtattgatggaggtg gaattagagg gatcattccg gctaccattc 300 tcgaatttct tgaaggacaacttcaggaaa tggacaataa tgcagatgca agacttgcag 360 attactttga tgtaattggaggaacaagta caggaggttt attgactgct atgataagta 420 ctccaaatga aaacaatcgaccctttgctg ctgccaaaga aattgtacct ttttacttcg 480 aacatggccc tcagatttttaatcctagtg gtcaaatttt aggcccaaaa tatgatggaa 540 aatatcttat gcaagttcttcaagaaaaac ttggagaaac tcgtgtgcat caagctttga 600 cagaagttgt catctcaagctttgacatca aaacaaataa gccagtaata ttcactaagt 660 caaatttagc aaactctccagaattggatg ctaagatgta tgacataagt tattccacag 720 cagcagctcc aacatattttcctccgcatt actttgttac taatactagt aatggagatg 780 aatatgagtt caatcttgttgatggtgctg ttgctactgt tgctgatccg gcgttattat 840 ccattagcgt tgcaacgagacttgcacaaa aggatccagc atttgcttca attaggtcat 900 tgaattacaa aaaaatgctgttgctctcat taggcactgg cactacttca gagtttgata 960 aaacatatac agcaaaagaggcagctacct ggactgctgt acattggatg ttagttatac 1020 agaaaatgac tgatgcagcaagttcttaca tgactgatta ttacctttct actgcttttc 1080 aagctcttga ttcaaaaaacaattacctca gggttcaaga ataatgag 1128 29 366 PRT synthetic Protein(1)..(366) Permutein protein encoded by pMON37408 29 Asn Ala Leu Thr GlyThr Thr Thr Glu Met Asp Asp Ala Ser Glu Ala 1 5 10 15 Asn Met Glu LeuLeu Val Gln Val Gly Glu Asn Leu Leu Lys Lys Pro 20 25 30 Val Ser Glu AspAsn Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe 35 40 45 Ala Lys Leu LeuSer Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser 50 55 60 Tyr Gly Pro GlyGln Leu Gly Glu Met Val Thr Val Leu Ser Ile Asp 65 70 75 80 Gly Gly GlyIle Arg Gly Ile Ile Pro Ala Thr Ile Leu Glu Phe Leu 85 90 95 Glu Gly GlnLeu Gln Glu Met Asp Asn Asn Ala Asp Ala Arg Leu Ala 100 105 110 Asp TyrPhe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly Leu Leu Thr 115 120 125 AlaMet Ile Ser Thr Pro Asn Glu Asn Asn Arg Pro Phe Ala Ala Ala 130 135 140Lys Glu Ile Val Pro Phe Tyr Phe Glu His Gly Pro Gln Ile Phe Asn 145 150155 160 Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp Gly Lys Tyr Leu Met165 170 175 Gln Val Leu Gln Glu Lys Leu Gly Glu Thr Arg Val His Gln AlaLeu 180 185 190 Thr Glu Val Val Ile Ser Ser Phe Asp Ile Lys Thr Asn LysPro Val 195 200 205 Ile Phe Thr Lys Ser Asn Leu Ala Asn Ser Pro Glu LeuAsp Ala Lys 210 215 220 Met Tyr Asp Ile Ser Tyr Ser Thr Ala Ala Ala ProThr Tyr Phe Pro 225 230 235 240 Pro His Tyr Phe Val Thr Asn Thr Ser AsnGly Asp Glu Tyr Glu Phe 245 250 255 Asn Leu Val Asp Gly Ala Val Ala ThrVal Ala Asp Pro Ala Leu Leu 260 265 270 Ser Ile Ser Val Ala Thr Arg LeuAla Gln Lys Asp Pro Ala Phe Ala 275 280 285 Ser Ile Arg Ser Leu Asn TyrLys Lys Met Leu Leu Leu Ser Leu Gly 290 295 300 Thr Gly Thr Thr Ser GluPhe Asp Lys Thr Tyr Thr Ala Lys Glu Ala 305 310 315 320 Ala Thr Trp ThrAla Val His Trp Met Leu Val Ile Gln Lys Met Thr 325 330 335 Asp Ala AlaSer Ser Tyr Met Thr Asp Tyr Tyr Leu Ser Thr Ala Phe 340 345 350 Gln AlaLeu Asp Ser Lys Asn Asn Tyr Leu Arg Val Gln Glu 355 360 365 30 1158 DNAsynthetic DNA (1)..(1158) pMON40701 sequence encoding permutein protein30 atggccacca ccaagagctt cctcatcctg atcttcatga tcctggccac caccagcagc 60accttcgccc agctcggcga gatggtgacc gtgctctcca tcgacggcgg tggcatcagg 120ggcatcatcc cggccaccat cctggagttc ctggagggcc aactccagga gatggacaac 180aacgccgacg cccgcctggc cgactacttc gacgtgatcg gtggcaccag caccggcggt 240ctcctgaccg ccatgatctc cactccgaac gagaacaacc gccccttcgc cgctgcgaag 300gagatcgtcc cgttctactt cgaacacggc cctcagattt tcaacccctc gggtcaaatc 360ctgggcccca agtacgacgg caagtacctt atgcaagtgc ttcaggagaa gctgggcgag 420actagggtgc accaggcgct gaccgaggtc gtcatctcca gcttcgacat caagaccaac 480aagccagtca tcttcaccaa gtccaacctg gccaacagcc cggagctgga cgctaagatg 540tacgacatct cctactccac tgctgccgct cccacgtact tccctccgca ctacttcgtc 600accaacacca gcaacggcga cgagtacgag ttcaaccttg ttgacggtgc ggtggctacg 660gtggcggacc cggcgctcct gtccatcagc gtcgccacgc gcctggccca gaaggatcca 720gccttcgcta gcattaggag cctcaactac aagaagatgc tgctgctcag cctgggcact 780ggcacgacct ccgagttcga caagacctac actgccaagg aggccgctac ctggaccgcc 840gtccattgga tgctggtcat ccagaagatg acggacgccg cttccagcta catgaccgac 900tactacctct ccactgcgtt ccaggcgctt gactccaaga acaactacct ccgtgttcag 960gagaatgccc tcactggcac cacgaccgag atggacgatg cctccgaggc caacatggag 1020ctgctcgtcc aggtgggtga gaacctcctg aagaagcccg tctccgaaga caatcccgag 1080acctatgagg aagcgctcaa gcgctttgcc aagctgctct ctgataggaa gaaactccgc 1140gctaacaagg ccagctac 1158 31 386 PRT synthetic Protein (1)..(386)Permutein protein encoded by pMON40701 sequence 31 Met Ala Thr Thr LysSer Phe Leu Ile Leu Ile Phe Met Ile Leu Ala 1 5 10 15 Thr Thr Ser SerThr Phe Ala Gln Leu Gly Glu Met Val Thr Val Leu 20 25 30 Ser Ile Asp GlyGly Gly Ile Arg Gly Ile Ile Pro Ala Thr Ile Leu 35 40 45 Glu Phe Leu GluGly Gln Leu Gln Glu Met Asp Asn Asn Ala Asp Ala 50 55 60 Arg Leu Ala AspTyr Phe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly 65 70 75 80 Leu Leu ThrAla Met Ile Ser Thr Pro Asn Glu Asn Asn Arg Pro Phe 85 90 95 Ala Ala AlaLys Glu Ile Val Pro Phe Tyr Phe Glu His Gly Pro Gln 100 105 110 Ile PheAsn Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp Gly Lys 115 120 125 TyrLeu Met Gln Val Leu Gln Glu Lys Leu Gly Glu Thr Arg Val His 130 135 140Gln Ala Leu Thr Glu Val Val Ile Ser Ser Phe Asp Ile Lys Thr Asn 145 150155 160 Lys Pro Val Ile Phe Thr Lys Ser Asn Leu Ala Asn Ser Pro Glu Leu165 170 175 Asp Ala Lys Met Tyr Asp Ile Ser Tyr Ser Thr Ala Ala Ala ProThr 180 185 190 Tyr Phe Pro Pro His Tyr Phe Val Thr Asn Thr Ser Asn GlyAsp Glu 195 200 205 Tyr Glu Phe Asn Leu Val Asp Gly Ala Val Ala Thr ValAla Asp Pro 210 215 220 Ala Leu Leu Ser Ile Ser Val Ala Thr Arg Leu AlaGln Lys Asp Pro 225 230 235 240 Ala Phe Ala Ser Ile Arg Ser Leu Asn TyrLys Lys Met Leu Leu Leu 245 250 255 Ser Leu Gly Thr Gly Thr Thr Ser GluPhe Asp Lys Thr Tyr Thr Ala 260 265 270 Lys Glu Ala Ala Thr Trp Thr AlaVal His Trp Met Leu Val Ile Gln 275 280 285 Lys Met Thr Asp Ala Ala SerSer Tyr Met Thr Asp Tyr Tyr Leu Ser 290 295 300 Thr Ala Phe Gln Ala LeuAsp Ser Lys Asn Asn Tyr Leu Arg Val Gln 305 310 315 320 Glu Asn Ala LeuThr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser Glu 325 330 335 Ala Asn MetGlu Leu Leu Val Gln Val Gly Glu Asn Leu Leu Lys Lys 340 345 350 Pro ValSer Glu Asp Asn Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg 355 360 365 PheAla Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala 370 375 380Ser Tyr 385 32 1167 DNA synthetic DNA (1)..(1167) pMON40703 sequenceencoding permutein protein 32 atggccacca ccaagagctt cctcatcctgatcttcatga tcctggccac caccagcagc 60 accttcgcca gcctcaacta caagaagatgctgctgctca gcctgggcac tggcacgacc 120 tccgagttcg acaagaccta cactgccaaggaggccgcta cctggaccgc cgtccattgg 180 atgctggtca tccagaagat gacggacgccgcttccagct acatgaccga ctactacctc 240 tccactgcgt tccaggcgct tgactccaagaacaactacc tccgtgttca ggagaatgcc 300 ctcactggca ccacgaccga gatggacgatgcctccgagg ccaacatgga gctgctcgtc 360 caggtgggtg agaacctcct gaagaagcccgtctccgaag acaatcccga gacctatgag 420 gaagcgctca agcgctttgc caagctgctctctgatagga agaaactccg cgctaacaag 480 gccagctacg gaccaggaca gctcggcgagatggtgaccg tgctctccat cgacggcggt 540 ggcatcaggg gcatcatccc ggccaccatcctggagttcc tggagggcca actccaggag 600 atggacaaca acgccgacgc ccgcctggccgactacttcg acgtgatcgg tggcaccagc 660 accggcggtc tcctgaccgc catgatctccactccgaacg agaacaaccg ccccttcgcc 720 gctgcgaagg agatcgtccc gttctacttcgaacacggcc ctcagatttt caacccctcg 780 ggtcaaatcc tgggccccaa gtacgacggcaagtacctta tgcaagtgct tcaggagaag 840 ctgggcgaga ctagggtgca ccaggcgctgaccgaggtcg tcatctccag cttcgacatc 900 aagaccaaca agccagtcat cttcaccaagtccaacctgg ccaacagccc ggagctggac 960 gctaagatgt acgacatctc ctactccactgctgccgctc ccacgtactt ccctccgcac 1020 tacttcgtca ccaacaccag caacggcgacgagtacgagt tcaaccttgt tgacggtgcg 1080 gtggctacgg tggcggaccc ggcgctcctgtccatcagcg tcgccacgcg cctggcccag 1140 aaggatccag ccttcgctag cattagg 116733 389 PRT synthetic Protein (1)..(389) Permutein protein encoded bypMON40703 sequence 33 Met Ala Thr Thr Lys Ser Phe Leu Ile Leu Ile PheMet Ile Leu Ala 1 5 10 15 Thr Thr Ser Ser Thr Phe Ala Ser Leu Asn TyrLys Lys Met Leu Leu 20 25 30 Leu Ser Leu Gly Thr Gly Thr Thr Ser Glu PheAsp Lys Thr Tyr Thr 35 40 45 Ala Lys Glu Ala Ala Thr Trp Thr Ala Val HisTrp Met Leu Val Ile 50 55 60 Gln Lys Met Thr Asp Ala Ala Ser Ser Tyr MetThr Asp Tyr Tyr Leu 65 70 75 80 Ser Thr Ala Phe Gln Ala Leu Asp Ser LysAsn Asn Tyr Leu Arg Val 85 90 95 Gln Glu Asn Ala Leu Thr Gly Thr Thr ThrGlu Met Asp Asp Ala Ser 100 105 110 Glu Ala Asn Met Glu Leu Leu Val GlnVal Gly Glu Asn Leu Leu Lys 115 120 125 Lys Pro Val Ser Glu Asp Asn ProGlu Thr Tyr Glu Glu Ala Leu Lys 130 135 140 Arg Phe Ala Lys Leu Leu SerAsp Arg Lys Lys Leu Arg Ala Asn Lys 145 150 155 160 Ala Ser Tyr Gly ProGly Gln Leu Gly Glu Met Val Thr Val Leu Ser 165 170 175 Ile Asp Gly GlyGly Ile Arg Gly Ile Ile Pro Ala Thr Ile Leu Glu 180 185 190 Phe Leu GluGly Gln Leu Gln Glu Met Asp Asn Asn Ala Asp Ala Arg 195 200 205 Leu AlaAsp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly Leu 210 215 220 LeuThr Ala Met Ile Ser Thr Pro Asn Glu Asn Asn Arg Pro Phe Ala 225 230 235240 Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe Glu His Gly Pro Gln Ile 245250 255 Phe Asn Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp Gly Lys Tyr260 265 270 Leu Met Gln Val Leu Gln Glu Lys Leu Gly Glu Thr Arg Val HisGln 275 280 285 Ala Leu Thr Glu Val Val Ile Ser Ser Phe Asp Ile Lys ThrAsn Lys 290 295 300 Pro Val Ile Phe Thr Lys Ser Asn Leu Ala Asn Ser ProGlu Leu Asp 305 310 315 320 Ala Lys Met Tyr Asp Ile Ser Tyr Ser Thr AlaAla Ala Pro Thr Tyr 325 330 335 Phe Pro Pro His Tyr Phe Val Thr Asn ThrSer Asn Gly Asp Glu Tyr 340 345 350 Glu Phe Asn Leu Val Asp Gly Ala ValAla Thr Val Ala Asp Pro Ala 355 360 365 Leu Leu Ser Ile Ser Val Ala ThrArg Leu Ala Gln Lys Asp Pro Ala 370 375 380 Phe Ala Ser Ile Arg 385 341167 DNA synthetic DNA (1)..(1167) pMON40705 sequence encoding permuteinprotein 34 atggccacca ccaagagctt cctcatcctg atcttcatga tcctggccaccaccagcagc 60 accttcgcca cctacactgc caaggaggcc gctacctgga ccgccgtccattggatgctg 120 gtcatccaga agatgacgga cgccgcttcc agctacatga ccgactactacctctccact 180 gcgttccagg cgcttgactc caagaacaac tacctccgtg ttcaggagaatgccctcact 240 ggcaccacga ccgagatgga cgatgcctcc gaggccaaca tggagctgctcgtccaggtg 300 ggtgagaacc tcctgaagaa gcccgtctcc gaagacaatc ccgagacctatgaggaagcg 360 ctcaagcgct ttgccaagct gctctctgat aggaagaaac tccgcgctaacaaggccagc 420 tacggaccag gacagctcgg cgagatggtg accgtgctct ccatcgacggcggtggcatc 480 aggggcatca tcccggccac catcctggag ttcctggagg gccaactccaggagatggac 540 aacaacgccg acgcccgcct ggccgactac ttcgacgtga tcggtggcaccagcaccggc 600 ggtctcctga ccgccatgat ctccactccg aacgagaaca accgccccttcgccgctgcg 660 aaggagatcg tcccgttcta cttcgaacac ggccctcaga ttttcaacccctcgggtcaa 720 atcctgggcc ccaagtacga cggcaagtac cttatgcaag tgcttcaggagaagctgggc 780 gagactaggg tgcaccaggc gctgaccgag gtcgtcatct ccagcttcgacatcaagacc 840 aacaagccag tcatcttcac caagtccaac ctggccaaca gcccggagctggacgctaag 900 atgtacgaca tctcctactc cactgctgcc gctcccacgt acttccctccgcactacttc 960 gtcaccaaca ccagcaacgg cgacgagtac gagttcaacc ttgttgacggtgcggtggct 1020 acggtggcgg acccggcgct cctgtccatc agcgtcgcca cgcgcctggcccagaaggat 1080 ccagccttcg ctagcattag gagcctcaac tacaagaaga tgctgctgctcagcctgggc 1140 actggcacga cctccgagtt cgacaag 1167 35 389 PRT syntheticProtein (1)..(389) Permutein protein encoded by pMON40705 35 Met Ala ThrThr Lys Ser Phe Leu Ile Leu Ile Phe Met Ile Leu Ala 1 5 10 15 Thr ThrSer Ser Thr Phe Ala Thr Tyr Thr Ala Lys Glu Ala Ala Thr 20 25 30 Trp ThrAla Val His Trp Met Leu Val Ile Gln Lys Met Thr Asp Ala 35 40 45 Ala SerSer Tyr Met Thr Asp Tyr Tyr Leu Ser Thr Ala Phe Gln Ala 50 55 60 Leu AspSer Lys Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala Leu Thr 65 70 75 80 GlyThr Thr Thr Glu Met Asp Asp Ala Ser Glu Ala Asn Met Glu Leu 85 90 95 LeuVal Gln Val Gly Glu Asn Leu Leu Lys Lys Pro Val Ser Glu Asp 100 105 110Asn Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe Ala Lys Leu Leu 115 120125 Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser Tyr Gly Pro Gly 130135 140 Gln Leu Gly Glu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly Ile145 150 155 160 Arg Gly Ile Ile Pro Ala Thr Ile Leu Glu Phe Leu Glu GlyGln Leu 165 170 175 Gln Glu Met Asp Asn Asn Ala Asp Ala Arg Leu Ala AspTyr Phe Asp 180 185 190 Val Ile Gly Gly Thr Ser Thr Gly Gly Leu Leu ThrAla Met Ile Ser 195 200 205 Thr Pro Asn Glu Asn Asn Arg Pro Phe Ala AlaAla Lys Glu Ile Val 210 215 220 Pro Phe Tyr Phe Glu His Gly Pro Gln IlePhe Asn Pro Ser Gly Gln 225 230 235 240 Ile Leu Gly Pro Lys Tyr Asp GlyLys Tyr Leu Met Gln Val Leu Gln 245 250 255 Glu Lys Leu Gly Glu Thr ArgVal His Gln Ala Leu Thr Glu Val Val 260 265 270 Ile Ser Ser Phe Asp IleLys Thr Asn Lys Pro Val Ile Phe Thr Lys 275 280 285 Ser Asn Leu Ala AsnSer Pro Glu Leu Asp Ala Lys Met Tyr Asp Ile 290 295 300 Ser Tyr Ser ThrAla Ala Ala Pro Thr Tyr Phe Pro Pro His Tyr Phe 305 310 315 320 Val ThrAsn Thr Ser Asn Gly Asp Glu Tyr Glu Phe Asn Leu Val Asp 325 330 335 GlyAla Val Ala Thr Val Ala Asp Pro Ala Leu Leu Ser Ile Ser Val 340 345 350Ala Thr Arg Leu Ala Gln Lys Asp Pro Ala Phe Ala Ser Ile Arg Ser 355 360365 Leu Asn Tyr Lys Lys Met Leu Leu Leu Ser Leu Gly Thr Gly Thr Thr 370375 380 Ser Glu Phe Asp Lys 385 36 10 PRT synthetic Protein (1)..(10)corn homolog peptide 36 Cys Ile Phe Asp Ser Thr Tyr Thr Ala Lys 1 5 1037 1161 DNA Solanum cardiophyllum exon (1)..(1161) patatin homolog Pat17nucleic acid and amino acid translation 37 atg gca act act aaa tct ttttta att tta ata ttt atg ata tta gca 48 Met Ala Thr Thr Lys Ser Phe LeuIle Leu Ile Phe Met Ile Leu Ala 1 5 10 15 act act agt tca aca ttt gctcag ttg gga gaa atg gtg act gtt ctt 96 Thr Thr Ser Ser Thr Phe Ala GlnLeu Gly Glu Met Val Thr Val Leu 20 25 30 agt att gat gga ggt gga att agaggg atc att ccg gct acc att ctc 144 Ser Ile Asp Gly Gly Gly Ile Arg GlyIle Ile Pro Ala Thr Ile Leu 35 40 45 gaa ttt ctt gaa gga caa ctt cag gaaatg gac aat aat gca gat gca 192 Glu Phe Leu Glu Gly Gln Leu Gln Glu MetAsp Asn Asn Ala Asp Ala 50 55 60 aga ctt gca gat tac ttt gat gta att ggagga aca agt aca gga ggt 240 Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly GlyThr Ser Thr Gly Gly 65 70 75 80 tta ttg act gct atg ata agt act cca aatgaa aac aat cga ccc ttt 288 Leu Leu Thr Ala Met Ile Ser Thr Pro Asn GluAsn Asn Arg Pro Phe 85 90 95 gct gct gcc aaa gaa att gta cct ttt tac ttcgaa cat ggc cct cag 336 Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe GluHis Gly Pro Gln 100 105 110 att ttt aat cct agt ggt caa att tta ggc ccaaaa tat gat gga aaa 384 Ile Phe Asn Pro Ser Gly Gln Ile Leu Gly Pro LysTyr Asp Gly Lys 115 120 125 tat ctt atg caa gtt ctt caa gaa aaa ctt ggagaa act cgt gtg cat 432 Tyr Leu Met Gln Val Leu Gln Glu Lys Leu Gly GluThr Arg Val His 130 135 140 caa gct ttg aca gaa gtt gtc atc tca agc tttgac atc aaa aca aat 480 Gln Ala Leu Thr Glu Val Val Ile Ser Ser Phe AspIle Lys Thr Asn 145 150 155 160 aag cca gta ata ttc act aag tca aat ttagca aac tct cca gaa ttg 528 Lys Pro Val Ile Phe Thr Lys Ser Asn Leu AlaAsn Ser Pro Glu Leu 165 170 175 gat gct aag atg tat gac ata agt tat tccaca gca gca gct cca aca 576 Asp Ala Lys Met Tyr Asp Ile Ser Tyr Ser ThrAla Ala Ala Pro Thr 180 185 190 tat ttt cct ccg cat tac ttt gtt act aatact agt aat gga gat gaa 624 Tyr Phe Pro Pro His Tyr Phe Val Thr Asn ThrSer Asn Gly Asp Glu 195 200 205 tat gag ttc aat ctt gtt gat ggt gct gttgct act gtt gct gat ccg 672 Tyr Glu Phe Asn Leu Val Asp Gly Ala Val AlaThr Val Ala Asp Pro 210 215 220 gcg tta tta tcc att agc gtt gca acg agactt gca caa aag gat cca 720 Ala Leu Leu Ser Ile Ser Val Ala Thr Arg LeuAla Gln Lys Asp Pro 225 230 235 240 gca ttt gct tca att agg tca ttg aattac aaa aaa atg ctg ttg ctc 768 Ala Phe Ala Ser Ile Arg Ser Leu Asn TyrLys Lys Met Leu Leu Leu 245 250 255 tca tta ggc act ggc act act tca gagttt gat aaa aca tat aca gca 816 Ser Leu Gly Thr Gly Thr Thr Ser Glu PheAsp Lys Thr Tyr Thr Ala 260 265 270 aaa gag gca gct acc tgg act gct gtacat tgg atg tta gtt ata cag 864 Lys Glu Ala Ala Thr Trp Thr Ala Val HisTrp Met Leu Val Ile Gln 275 280 285 aaa atg act gat gca gca agt tct tacatg act gat tat tac ctt tct 912 Lys Met Thr Asp Ala Ala Ser Ser Tyr MetThr Asp Tyr Tyr Leu Ser 290 295 300 act gct ttt caa gct ctt gat tca aaaaac aat tac ctc agg gtt caa 960 Thr Ala Phe Gln Ala Leu Asp Ser Lys AsnAsn Tyr Leu Arg Val Gln 305 310 315 320 gaa aat gca tta aca ggc aca actact gaa atg gat gat gct tct gag 1008 Glu Asn Ala Leu Thr Gly Thr Thr ThrGlu Met Asp Asp Ala Ser Glu 325 330 335 gct aat atg gaa tta tta gta caagtt ggt gaa aac tta ttg aag aaa 1056 Ala Asn Met Glu Leu Leu Val Gln ValGly Glu Asn Leu Leu Lys Lys 340 345 350 cca gtt tcc gaa gac aat cct gaaacc tat gag gaa gct cta aag agg 1104 Pro Val Ser Glu Asp Asn Pro Glu ThrTyr Glu Glu Ala Leu Lys Arg 355 360 365 ttt gca aaa ttg ctc tct gat aggaag aaa ctc cga gca aac aaa gct 1152 Phe Ala Lys Leu Leu Ser Asp Arg LysLys Leu Arg Ala Asn Lys Ala 370 375 380 tct tat taa 1161 Ser Tyr 385 381158 DNA Solanum tuberosum DNA (1)..(1158) DNA sequence encoding apatatin (acyl lipid hydrolase) protein 38 atggcaacta ctaaatcttttttaatttta atatttatga tattagcaac tactagttca 60 acatttgctc agttgggagaaatggtgact gttcttagta ttgatggagg tggaattaga 120 gggatcattc cggctaccattctcgaattt cttgaaggac aacttcagga aatggacaat 180 aatgcagatg caagacttgcagattacttt gatgtaattg gaggaacaag tacaggaggt 240 ttattgactg ctatgataagtactccaaat gaaaacaatc gaccctttgc tgctgccaaa 300 gaaattgtac ctttttacttcgaacatggc cctcagattt ttaatcctag tggtcaaatt 360 ttaggcccaa aatatgatggaaaatatctt atgcaagttc ttcaagaaaa acttggagaa 420 actcgtgtgc atcaagctttgacagaagtt gtcatctcaa gctttgacat caaaacaaat 480 aagccagtaa tattcactaagtcaaattta gcaaactctc cagaattgga tgctaagatg 540 tatgacataa gttattccacagcagcagct ccaacatatt ttcctccgca ttactttgtt 600 actaatacta gtaatggagatgaatatgag ttcaatcttg ttgatggtgc tgttgctact 660 gttgctgatc cggcgttattatccattagc gttgcaacga gacttgcaca aaaggatcca 720 gcatttgctt caattaggtcattgaattac aaaaaaatgc tgttgctctc attaggcact 780 ggcactactt cagagtttgataaaacatat acagcaaaag aggcagctac ctggactgct 840 gtacattgga tgttagttatacagaaaatg actgatgcag caagttctta catgactgat 900 tattaccttt ctactgcttttcaagctctt gattcaaaaa acaattacct cagggttcaa 960 gaaaatgcat taacaggcacaactactgaa atggatgatg cttctgaggc taatatggaa 1020 ttattagtac aagttggtgaaaacttattg aagaaaccag tttccgaaga caatcctgaa 1080 acctatgagg aagctctaaagaggtttgca aaattgctct ctgataggaa gaaactccga 1140 gcaaacaaag cttcttat1158 39 386 PRT potato Protein (1)..(386) potato patatin proteinsequence 39 Met Ala Thr Thr Lys Ser Phe Leu Ile Leu Ile Phe Met Ile LeuAla 1 5 10 15 Thr Thr Ser Ser Thr Phe Ala Gln Leu Gly Glu Met Val ThrVal Leu 20 25 30 Ser Ile Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala ThrIle Leu 35 40 45 Glu Phe Leu Glu Gly Gln Leu Gln Glu Met Asp Asn Asn AlaAsp Ala 50 55 60 Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser ThrGly Gly 65 70 75 80 Leu Leu Thr Ala Met Ile Ser Thr Pro Asn Glu Asn AsnArg Pro Phe 85 90 95 Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe Glu HisGly Pro Gln 100 105 110 Ile Phe Asn Pro Ser Gly Gln Ile Leu Gly Pro LysTyr Asp Gly Lys 115 120 125 Tyr Leu Met Gln Val Leu Gln Glu Lys Leu GlyGlu Thr Arg Val His 130 135 140 Gln Ala Leu Thr Glu Val Val Ile Ser SerPhe Asp Ile Lys Thr Asn 145 150 155 160 Lys Pro Val Ile Phe Thr Lys SerAsn Leu Ala Asn Ser Pro Glu Leu 165 170 175 Asp Ala Lys Met Tyr Asp IleSer Tyr Ser Thr Ala Ala Ala Pro Thr 180 185 190 Tyr Phe Pro Pro His TyrPhe Val Thr Asn Thr Ser Asn Gly Asp Glu 195 200 205 Tyr Glu Phe Asn LeuVal Asp Gly Ala Val Ala Thr Val Ala Asp Pro 210 215 220 Ala Leu Leu SerIle Ser Val Ala Thr Arg Leu Ala Gln Lys Asp Pro 225 230 235 240 Ala PheAla Ser Ile Arg Ser Leu Asn Tyr Lys Lys Met Leu Leu Leu 245 250 255 SerLeu Gly Thr Gly Thr Thr Ser Glu Phe Asp Lys Thr Tyr Thr Ala 260 265 270Lys Glu Ala Ala Thr Trp Thr Ala Val His Trp Met Leu Val Ile Gln 275 280285 Lys Met Thr Asp Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser 290295 300 Thr Ala Phe Gln Ala Leu Asp Ser Lys Asn Asn Tyr Leu Arg Val Gln305 310 315 320 Glu Asn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp AlaSer Glu 325 330 335 Ala Asn Met Glu Leu Leu Val Gln Val Gly Glu Asn LeuLeu Lys Lys 340 345 350 Pro Val Ser Glu Asp Asn Pro Glu Thr Tyr Glu GluAla Leu Lys Arg 355 360 365 Phe Ala Lys Leu Leu Ser Asp Arg Lys Lys LeuArg Ala Asn Lys Ala 370 375 380 Ser Tyr 385 40 452 PRT synthetic Protein(1)..(452) Pre-cleavage patatin protein produced in Pichia pastoris 40Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 1015 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 2530 Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 4045 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 5560 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 7075 80 Ser Leu Glu Lys Arg Glu Ala Glu Ala Gln Leu Gly Glu Met Val Thr 8590 95 Val Leu Ser Ile Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr100 105 110 Ile Leu Glu Phe Leu Glu Gly Gln Leu Gln Glu Met Asp Asn AsnAla 115 120 125 Asp Ala Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly ThrSer Thr 130 135 140 Gly Gly Leu Leu Thr Ala Met Ile Ser Thr Pro Asn GluAsn Asn Arg 145 150 155 160 Pro Phe Ala Ala Ala Lys Glu Ile Val Pro PheTyr Phe Glu His Gly 165 170 175 Pro Gln Ile Phe Asn Pro Ser Gly Gln IleLeu Gly Pro Lys Tyr Asp 180 185 190 Gly Lys Tyr Leu Met Gln Val Leu GlnGlu Lys Leu Gly Glu Thr Arg 195 200 205 Val His Gln Ala Leu Thr Glu ValVal Ile Ser Ser Phe Asp Ile Lys 210 215 220 Thr Asn Lys Pro Val Ile PheThr Lys Ser Asn Leu Ala Asn Ser Pro 225 230 235 240 Glu Leu Asp Ala LysMet Tyr Asp Ile Ser Tyr Ser Thr Ala Ala Ala 245 250 255 Pro Thr Tyr PhePro Pro His Tyr Phe Val Thr Asn Thr Ser Asn Gly 260 265 270 Asp Glu TyrGlu Phe Asn Leu Val Asp Gly Ala Val Ala Thr Val Ala 275 280 285 Asp ProAla Leu Leu Ser Ile Ser Val Ala Thr Arg Leu Ala Gln Lys 290 295 300 AspPro Ala Phe Ala Ser Ile Arg Ser Leu Asn Tyr Lys Lys Met Leu 305 310 315320 Leu Leu Ser Leu Gly Thr Gly Thr Thr Ser Glu Phe Asp Lys Thr Tyr 325330 335 Thr Ala Lys Glu Ala Ala Thr Trp Thr Ala Val His Trp Met Leu Val340 345 350 Ile Gln Lys Met Thr Asp Ala Ala Ser Ser Tyr Met Thr Asp TyrTyr 355 360 365 Leu Ser Thr Ala Phe Gln Ala Leu Asp Ser Lys Asn Asn TyrLeu Arg 370 375 380 Val Gln Glu Asn Ala Leu Thr Gly Thr Thr Thr Glu MetAsp Asp Ala 385 390 395 400 Ser Glu Ala Asn Met Glu Leu Leu Val Gln ValGly Glu Asn Leu Leu 405 410 415 Lys Lys Pro Val Ser Glu Asp Asn Pro GluThr Tyr Glu Glu Ala Leu 420 425 430 Lys Arg Phe Ala Lys Leu Leu Ser AspArg Lys Lys Leu Arg Ala Asn 435 440 445 Lys Ala Ser Tyr 450 41 367 PRTsynthetic Protein (1)..(367) Post-cleavage patatin protein produced inPichia pastoris 41 Glu Ala Glu Ala Gln Leu Gly Glu Met Val Thr Val LeuSer Ile Asp 1 5 10 15 Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr IleLeu Glu Phe Leu 20 25 30 Glu Gly Gln Leu Gln Glu Met Asp Asn Asn Ala AspAla Arg Leu Ala 35 40 45 Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr GlyGly Leu Leu Thr 50 55 60 Ala Met Ile Ser Thr Pro Asn Glu Asn Asn Arg ProPhe Ala Ala Ala 65 70 75 80 Lys Glu Ile Val Pro Phe Tyr Phe Glu His GlyPro Gln Ile Phe Asn 85 90 95 Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr AspGly Lys Tyr Leu Met 100 105 110 Gln Val Leu Gln Glu Lys Leu Gly Glu ThrArg Val His Gln Ala Leu 115 120 125 Thr Glu Val Val Ile Ser Ser Phe AspIle Lys Thr Asn Lys Pro Val 130 135 140 Ile Phe Thr Lys Ser Asn Leu AlaAsn Ser Pro Glu Leu Asp Ala Lys 145 150 155 160 Met Tyr Asp Ile Ser TyrSer Thr Ala Ala Ala Pro Thr Tyr Phe Pro 165 170 175 Pro His Tyr Phe ValThr Asn Thr Ser Asn Gly Asp Glu Tyr Glu Phe 180 185 190 Asn Leu Val AspGly Ala Val Ala Thr Val Ala Asp Pro Ala Leu Leu 195 200 205 Ser Ile SerVal Ala Thr Arg Leu Ala Gln Lys Asp Pro Ala Phe Ala 210 215 220 Ser IleArg Ser Leu Asn Tyr Lys Lys Met Leu Leu Leu Ser Leu Gly 225 230 235 240Thr Gly Thr Thr Ser Glu Phe Asp Lys Thr Tyr Thr Ala Lys Glu Ala 245 250255 Ala Thr Trp Thr Ala Val His Trp Met Leu Val Ile Gln Lys Met Thr 260265 270 Asp Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser Thr Ala Phe275 280 285 Gln Ala Leu Asp Ser Lys Asn Asn Tyr Leu Arg Val Gln Glu AsnAla 290 295 300 Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser Glu AlaAsn Met 305 310 315 320 Glu Leu Leu Val Gln Val Gly Glu Asn Leu Leu LysLys Pro Val Ser 325 330 335 Glu Asp Asn Pro Glu Thr Tyr Glu Glu Ala LeuLys Arg Phe Ala Lys 340 345 350 Leu Leu Ser Asp Arg Lys Lys Leu Arg AlaAsn Lys Ala Ser Tyr 355 360 365 42 7 PRT synthetic Protein (1)..(7) Xaa3= Phe, Ile, or Leu; Xaa5 = His or Asn 42 Phe Tyr Xaa Glu Xaa Gly Pro 1 543 55 DNA synthetic DNA (1)..(55) oligonucleotide-3 43 ggagctcgagaaaagagagg ctgaagcttc attgaattac aaaaaaatgc tgttg 55 44 42 DNA syntheticDNA (1)..(42) oligonucleotide-4 44 tcccaactgt cctggtccat aagaagctttgtttgctcgg ag 42 45 36 DNA synthetic DNA (1)..(36) oligonucleotide-5 45gcttcttatg gaccaggaca gttgggagaa atggtg 36 46 39 DNA synthetic DNA(1)..(39) oligonucleotide-6 46 ggtctagagg aattctcatt acctaattgaagcaaatgc 39 47 39 DNA synthetic DNA (1)..(39) oligonucleotide-7 47ggtctagagg aattctcatt aagtaacaaa gtaatgcgg 39 48 55 DNA synthetic DNA(1)..(55) oligonucleotide-8 48 ggagctcgag aaaagagagg ctgaagctaatactagtaat ggagatgaat atgag 55 49 55 DNA synthetic DNA (1)..(55)oligonucleotide-9 49 ggagctcgag aaaagagagg ctgaagctag ttattccacagcagcagctc caaca 55 50 39 DNA synthetic DNA (1)..(39) oligonucleotide-1050 ggtctagagg aattctcatt atatgtcata catcttagc 39 51 55 DNA synthetic DNA(1)..(55) oligonucleotide-11 51 ggagctcgag aaaagagagg ctgaagctacatatacagca aaagaggcag ctacc 55 52 39 DNA synthetic DNA (1)..(39)oligonucleotide-12 52 ggtctagagg aattctcatt atttatcaaa ctctgaagt 39 5355 DNA synthetic DNA (1)..(55) oligonucleotide-13 53 ggagctcgagaaaagagagg ctgaagctaa tgcattaaca ggcacaacta ctgaa 55 54 39 DNA syntheticDNA (1)..(39) oligonucleotide-14 54 ggtctagagg aattctcatt attcttgaaccctgaggta 39 55 55 DNA synthetic DNA (1)..(55) oligonucleotide-15 55ggagctcgag aaaagagagg ctgaagctag cctcaactac aagaagatgc tgctg 55 56 42DNA synthetic DNA (1)..(42) oligonucleotide-16 56 gccgagctgt cctggtccgtagctggcctt gttagcgcgg ag 42 57 36 DNA synthetic DNA (1)..(36)oligonucleotide-17 57 gccagctacg gaccaggaca gctcggcgag atggtg 36 58 39DNA synthetic DNA (1)..(39) oligonucleotide-18 58 ggtctagagg aattctcattacctaatgct agcgaaggc 39 59 55 DNA synthetic DNA (1)..(55)oligonucleotide-19 59 ggagctcgag aaaagagagg ctgaagctac tgccaaggaggccgctacct ggacc 55 60 39 DNA synthetic DNA (1)..(39) oligonucleotide-2060 ggtctagagg aattctcatt acttgtcgaa ctcggaggt 39

1-7 (canceled)
 8. A method for protecting a plant from Coleopteraninsect infestation comprising providing to said plant a Coleopteraninsect inhibitory amount of a protein exhibiting lipid acyl hydrolaseactivity, wherein said protein comprises a) a first motif comprisingGly-Xaa₁-Ser-Xaa₂-Gly as set forth in SEQ ID NO:14, wherein Xaa₁ andXaa₂ are Ser or Thr; b) a second motif comprisingGlu-Xaa₁-Xaa₂-Leu-Val-Asp-Gly as set forth in SEQ ID NO:15, wherein Xaa₁comprises the amino acids selected from the group consisting of Tyr,Phe, and Trp, and wherein Xaa₂ comprises the amino acids selected fromthe group consisting of His and Asn; and c) a third motif comprisingPhe-Tyr-Xaa₁-Glu-Xaa₂-Gly-Pro as set forth in SEQ ID NO:42, wherein Xaa₁comprises the amino acids selected from the group consisting of Phe,Ile, and Leu, and wherein Xaa₂ comprises the amino acids selected fromthe group consisting of His and Asn.
 9. The method according to claim 8wherein said protein is selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:40, and SEQID NO:41.
 10. The method according to claim 8 wherein said protein isnot naturally occurring. 11-12 (Canceled)